Chapter I
Introduction
Plant and human are inseparable because plants not only provide with food, shelter and medicine but also the life sustaining oxygen gas. Since disease, decay and death have always co-existed with life, the early man had to think about disease and its treatment at the dawn of human intellect. Medicinal plants represent a rich source from which antimicrobial agents may be obtained. Plants are used medicinally in different countries and are source of many potent and powerful drugs. The active principles of many drugs found in plants are secondary metabolites (Ghani 2003). There, basic phytochemical investigation of these extracts for their major phyto constituents is also vital. The natural products derived from medicinal plants have proven to be an abundant source of biologically active compounds, many of which have been the basis for the development of new lead chemicals for pharmaceuticals. With respect to infectious diseases, the increasing resistance in many common pathogens to currently used therapeutic agents, such as antibiotics and antiviral, has led to renewed interest in the discovery of novel anti-infective compounds (Groombridge and Jenkins, 2002). Over the years, medicinal plants have been found useful in the treatment and management of various health problems. About 80% of the world population relies on the use of traditional medicine, which is predominantly based on plant material (WHO, 1993). Scientific studies available on a good number of medicinal plants indicate that promising phytochemicals can be developed for many health problems (Gupta, 1994). Herbal drugs have gained importance in recent years because of their efficacy and cost effectiveness. These drugs are invariably single plant extracts of fractions thereof or mixtures of fractions/extracts from different plants, which have been carefully standardized for their safety and efficacy (Subramoniam and Pushpangadan, 1999). Mangifera indica L. (Anacardiaceae) is one of the most important tropical plants marketed in the world (Ross, 1999). It is a large tree that grows in tropical and subtropical regions, whose fruits are widely appreciated by the population. There are many traditional medicinal uses for the bark, roots and leaves of M. indica throughout the globe (Ross, 1999).
This species is purported to posses numerous therapeutic uses including analgesic, anti- inflammatory (Garrido et al., 2001), immunostimulant (Garcia et al., 2002), antioxidant (Martinez et al., 2000), spasmolytic, antidiarrhea (Sairam et al., 2003), dyslipidemic (Anila et al., 2002)],antidiabetic (Aderibigbe et al., 1999), antiamebic(Tona et al.,2000),
anthelminthic, antiallergic (Garcia et al., 2003) and antibacterial applications(Bairy et al., 2002). Therefore, the study aimed to evaluate the antidiarrhoeal activity of the alcoholic extraction obtained from the leaves of Mangifera indica using different in vivo experimental models in rodents followed by its antimicrobial investigation.
Chapter II
Review of literature
Charu Gupta et al., 2009, conducted a study for antimicrobial activity of dry mango pulp called amchur (dried pulp of unripe Mangifera indica) extract (50% ethanol) was tested against ten bacterial strains (7 Gram-positive and 3 Gram-negative) by agar well diffusion assays. The crude extract exhibited a broad spectrum of antibacterial activity inhibiting both the groups of bacteria. The extract was most effective against Staphylococcus aureus (26.0mm). Bacillus mycoides was found to be the most sensitive, with the lowest MIC of 62.5mg/mL, followed by Staphylococcus aureus and Pseudomonas aeruginosa (125mg/mL each), whereas Micrococcus luteus was found to be the most resistant surviving up to 500mg/mL. However, Only Alternaria sp. & Penicillium sp. was found to be partially sensitive to the extract.
Rames C. et al., 2009, carried out a research work for phytochemical analysis of amchur extract revealed the presence of tannins & terpenes. This study shows the potential for replacement of synthetic preservatives by the use of natural extracts.
Ayoola et al., 2008 , in their research syudy concluded the result of phytochemical screening showed that the leaves of Mangifera indica contain flavonoids, terpenoids,saponins, tannins and reducing sugars but did not contain cardiac glycosides and alkaloids.
Doughari and Manzara , 2008, reveled that the result of active components of leaves of Mangifera indica L. were extracted using cold water and organicsolvents (acetone and methanol) and were tested against Staphylococcus aureus, Streptococcus, pyogenase, Streptococcus pneumoniae, Bacillus cereus, Escherichia coli, Pseudomonas aerugenosa, Proteus mirabilis, Salmonella typhi and Shigella flexnerri using the agar well (cup plate) diffusion method. Both the acetone and methanol extracts inhibited the growth of gram positive bacteria, with acetone extract exerting more activities on all the gram positive bacteria with zone of inhibition between 15 - 16 mm, and a gram negative bacterium S.
typhi (14 mm) at 250 mg/ml. Whereas, water extract was not active on any of the bacterial pathogens tested at any of the concentration of the extract used. The activities of the plant extracts on the inhibited pathogens using the zone of inhibition were not as effective as the standard commercial antibacterial disks of gentamicin and erythromycin (t =2.23, p
<0.05). Increased temperature (60 and 100°C for 1 h) had a multiplier effect on the
activity of the extracts, but alkaline pH decreased the activity. Preliminary phytochemical analysis revealed the presence oftannins, glycosides, saponins and phenols.
Juliana et al ., 2009, was designed to determine the gastroprotective effect of a Mangifera indica leaf decoction (AD), on different experimental models in rodents. The administration of AD up to a dose of 5 g/kg (p.o.) did not produce any signs or symptoms of toxicity in the treated animals, while significantly decreasing the severity of gastric damage induced by several gastroprotective models.
According to K.Sairam et al., 2002, Mangifera indica is commonly grown in many parts of the world. Its seeds have been used for anti-diarrhoeal activity in Indian traditional medicine. Their study evaluates the potential anti-diarrhoeal activity of methanolic(MMI) and aqueous (AMI) extracts of seeds of M. indica in experimental diarrhoea, induced by castor oil and magnesium sulphate in mice. Both MMI and AMI were given orally in the dose of 250 mg/kg, showed significant anti-diarrhoeal activity comparable with that of the standard drug loperamide. However ,MMI significantly reduced intestinal transit in charcoal meal test as compared with atropine sulphate (5mg/kg; im).
Rosan et al., 1992 , a randomized placebo-controlled trials proved that plant leaf extract have (Mangifera indica) cardiotoxicity.
Ashok kumar et al.,2002 ,conducted the in vitro antimicrobial activity of MMI and AMI showed variable results. While, AMI significantly inhibited growth of Streptococcus aureus and Proteus vulgaris , both MMI and AMI did not show any significant effect on growth of E. coli and Klebsiella . The results illustrate that the extracts of M. indica have significant anti-diarrhoeal activity and part of the activity of MMI may be attributed to its effect on intestinal transit.
Lin SC et al., 1996, revealed that the protective effect of Mangifera indica R. Br. on hepatotoxin-induced acute liver damage.
Khan MR et al., 2003, study the Antibacterial activity of Mangifera indica .
Jagetia GC, et al., 2003, induction of developmental toxicity in mice treated with Mangifera indica (mango) In utero.
Baliga MS, et al., 2004, conducted the evaluation of the acute toxicity and long term safety of hydroalcoholic extract of Mango (Mangifera indica) in mice and rats.
Chapter III
Materials and Methods
Animals and diet:
Male albino rats obtained from the animal house of BCSIR laboratory, Chittagong weighing 180-200 gm were used in the entire study. The animals were acclimatized to standard laboratory conditions (Temperature 24± 1ºC, relative humidity 55 ±5% and 12 h photoperiod) in suspended wire-meshed galvanized cages (4/6 rats/cage) for one week before the commencement of the experiment. During the entire period of study, the rats were supplied with semi-purified basal diet and adlibitum. All animals were maintained according to the NIH guidelines of care and use of laboratory animals published by Saha et al., 2001.
Preparation of plant Extracts:
The plan leaves of Mangifera indica were collected from the BCSIR Laboratories;
Chittagong Campus; Chittagong with the help of industrial Botany Research Division. The collected samples were cleaned; air-dried and ground into powder. The dried powder 500g was extracted with ethanol. The extracts were filtered and the filtrates were evaporated to dryness at 40ºC under vacuum and finally freeze dried to get ethanol (63.85g) extract.
Castor oil induced diarrhea:
The method of Niemeegers et al., (1976) as adopted by Yaegnanarayan et al., (1982) was followd. Rats weighing 150-200gm were employed for this study. They were all screened initially by giving 2.0ml of castor oil orally and only those showing diarrhoea were were selected for further study. Extract pre-treatment was given orally 1 hour before the rats were administered with the standard dose for 2.0 ml castor oil. The animals were caged individually and examined for the presence of diarrhea ½ hourly for 6 hours after the castor oil challenge. Diarrhoea was defined as the presence in the stool of fluid material that stained the absorbent placed beneath the cage. The number of respondents and the number of stools passed during the 6-hour period was noted for each rat.The percent (%) inhibition of defecation was measured using the following formula.
% Inhibition of defecation = [(A - B) / A] x 100 A = Mean number of defecation caused by castor oil B = Mean number of defecation caused by drug or extract
Bacterial test organism:
Alcholic extacts of Mangifera indica were tested for their antibactarial activity against various gram- negative and gram-positive organisms, such as Bacillus subtilis (BTCC 17), B. megaterium (BTCC 18) B. cereus (BTCC 19), Staphylococcus aureus (ATCC6538), Salmonella typhi (AE14612), Shigella flexinary (ICDDR’B), Shigella sonnei [CRL (ICDDR, B)], Klebsiella sp[CRL (ICDDR’ B)], Proteus sp[CRL (ICDDR’ B)], Bacillus polimyxa (BTCC 16). These bacterial test organisms were collected from Bangladesh Type Culture Center, Institute of Nutrition and Food Science, Dhaka University and ICDDR, B.
Disc diffusion method:
The bioassay for bacterial strains was employed by disc diffusion method (Ergene et al., 2006). Filter paper discs (Whatman No. 1) of 5 mm diameter were loaded with crude extracts. Discs were completely dried and sterilized. 100 μl of cultures were spread on Sterilized nutrient agar media; impregnated discs were placed on it and incubated for 24 hrs at 37ºC. Tetracycline discs (30 μg/disc) were used as a standard drug. The diameter of zone of inhibition in mm was recorded after incubation. The experiment was performed intriplicates and average diameter of zone of inhibition was obtained
Cytotoxicity assay:
The cytotoxicity assay was performed on brine shrimp nauplii using Mayer method. Brine shrimp nauplii were obtained by hatching brine shrimp eggs (Carolina Biological Company, Burlington, NC, USA) in artificial sea-water (22.5 gm sodium chloride dissolved in 500 ml distilled water) for 48 hours. Dissolution of extract was performed in artificial sea-water by using twine. Each 5 ml solution of different concentrations (62.5, 125, 250, 500, 1000, 2000 µg/ml) of the extract was taken in different beakers where brine shrimp nauplii were given and observed for mortality for 24 hours. The resulting data were transformed to probity analysis for the determination of LC50 values of the extract.
Artificial sea-water and artificial sea-water medium Containing twine both were used as control.
For the hatching of brine shrimp nauplii following instructions (Artemia cysts) were obeyed;
1. Tank: Cone shaped for optimum circulation
2. Aeration: Sufficient aeration at the bottom of the tank was needed in order to keep the brine shrimp eggs circulating and in suspension at all.
3. Solution: Clean natural sea water was used and dilute and dilute 15-25ppt or prepared a hatching solution using non ionized salt and tap water.
4. Optimum condition: PH: 8.1-8.4
Cyst density:2-3g/L (Average 1-2g/L) Salinity:20-35g/L
Temparature:27-29ºC or 80-85ºF
[Temperature above 30ºC was always avoided].
5. Light: Natural sunlight or artificial light of 2000lux needed during the first 12hrs of hatching for maximum yield.
6. Hatching time: A complete hatch was achieved in 16-20hrs following the hatching instructions.
For longer self life, stored in dry place at a temperature below 20ºC.
Hatching:
Hydrate cysts for 1hr in fresh water to shorten the hatching time.
Rinsed hydrated cysts & start hatch
When harvesting concentrate nauplii at bottom of tank by turning off lights and aeration
Statistical Analysis:
Data were expressed as Mean±S.E. (StandardError). Unpaired ‘t’ tests were done for significant tests. SPSS for WINDOWS@TM Statistical Package for social science for Windows was applied for all analyses of data. Probability (P) value of 0.05 or less (P≤0.05) was considered as significant. In case of cytotoxicity , the LC50 valued were calculated by “Probit Analysis”. In this case “Bio-State Software-2007” was used for probit analysis and Chi-sqare test.
Fig 1: Shed dried leaves sample of Mangifera indica
Fig 2 : Grinder
Fig 3: Extraction process of Mangifera indica
Fig 4 : Alocoholic dipping of dried leaves (Mangifera indica)
Preparation Of Mangifera indica Leaves Extract.
Antimicrobial, Anti-diarrhoeal And Cytotoxicity Activity Test.
Fig 5: Anti-microbial test Mangifera indica
Fig 8 : Anti microbial test on dried diffusion method.
Fig 6: Anti diarrhoeal test in rats.
Fig 8 Different concentration of brine shrimp larvae.
Chapter IV
Result and discussion Antibacterial Assay of Mango (Mangifera indica
) Result of antibacterial effect:Table: 1 Antimicrobial activity against ten pathogenic bacteria Bacterial culture Diameter of zone of inhibition(mm)
Alcoholic extract of mango (100µg/disc)
Tetraclycine (30 µg/disc)
Bacillus subtilis 8 10
Bacillus cereus 10.5 10.5
Bacillus megaterium 10.5 12
Saphylococcus aureus 9.5 16.5
Shigella flexinary 7.5 7
Klebsiella sp. - 28
Salmonella typhi - 14.5
Shigella somnei 7.5 7.5
Proteus sp. 12.5 -
Bacillus polimyxa 8 15.5
“ - “indicates no activity
Discussion:
The alchoholic extracts of Mangifera indica were tested for antibacterial activities against 10 bacterial test organisms (Table 1).The extracts shows prominent antibacterial activity against eight bacteria species- Bacillus subtilis (BTCC 17), B. megaterium (BTCC 18) B.
cereus (BTCC 19), Staphylococcus aureus (ATCC6538), Shigella flexinary[CRL(ICDD’
B)], Shigella sonnei [ CRL (ICDDR, B)] , Proteus sp[CRL(ICDDR’ B)] and Bacillus polimyxa(BTCC 16). Among them Proteus sp[CRL(ICDDR’ B)] culture exhibited the
largest zone of inhibition (12.5 mm).There was no antibacterial activity against Klebsiella sp[CRL(ICDDR’ B)], Salmonell typhi[CRL(ICDDR’ B)] test organism. The extract from the leaves of mango, contains polyphenols whose major ingredient is mangiferin, [C- glucosylxanthone (1, 3, 6, 7-tetrahydroxyxanthone-C2-β-D-glucoside)] (Ho. L, 2003). The solutions of mangiferin in polyethylene glycol-400 showed an activity with regard to seven bacterial- Bacillus pumilus, Bacillus cereus, Staphylococcus aureus, Staphylococcus citreus, Escherichia coli, Salmonella agona, Klebsiella pneumoniae,in the earlier study.( I.
Stoilova et al.,2005)
Antidiarrhoel Assay of Mango(Mangifera indica)
Result of antidiarrhoel effect :
In the castor oil-induced diarrhoea experiment, the rat group that did not received the plant extract showed typical diarrhoeal signs and symptoms such as watery and frequent defecation. The extract of Mangifera indica produced a notable antidiarrhoeal effect in rat.
(Table-4)The crude extract significantly decreased (p < 0.01) the total number of wet Fig 9 : Zone of inhibition produced by Mangifera
indica .
faeces produced by administration of castor oil (2.0 ± 0.84) at the dose of 200 mg/kg as compared to the castor oil-treated control group (7.2±0.49) at sixth hour of observation.
The percentage of inhibition of castor oil-induced diarrhoea in the extract-treated rat was 72.2%.The effect of the extract was found to be close to that of the standard drug, loperamide (2 mg/kg), which produced an inhibition of 75% (Table-4)
Group Treatment Dose Number of
rats with diarrhoea
% Protectio
n
Mean defecation within 6 hrs
study
% Inhibit
ion of defecti
on
Control Distilled water
2 ml/rat 5/5 0 %
7.2 ± 0.49
-
Positive
control Loperamide 2 mg/kg 3/5 40 % 1.8 ± 1.11 75 %
Treated Ethanol Extract of M. indica
200 mg/kg
3/5 40 % 2.0 ± 0.84 72.2%
Table 2: Effect of ethanol extract of M. indica leaves on castor oil induced diarrhoea.
Table 3: Comparing the effect of M. indica and loperamadie on mean defecation of albino rats using Student’s‘t’ test.
,
Mean defecation (6 hrs study period)
Control Loperamide
(2 mg/kg) M. indica leaves extract (200mg/kg)
7.2 ± 0.49 1.8 ± 1.11** 2.0 ± 0.84**
S tu d en t’ s ‘t ’t es t
t calculated t tabulated
degrees of freedom P value
4.44 3.35 8.0
<0.01
5.36 3.35 8.0
<0.01
All values are expressed as mean±SEM (n=5). **P<0.01 significant compared to control (Student’s ‘t’ test)
Table 4 : Antidiarrhoeal effect of Mango leaves on castor oil induce diarrhea
Group Treatment Latent time
(Min)
Number of feces at first hour
Number of watery feces at sixth hour Hard
Stool
Watery feces
Control Castor oil(2 ml/rat) 12.90±0.43 8.20±0.48 3.0 ±0.32 7.2 ± 0.49 Standard Cator oil +
Loperamide) 2 mg/kg
39.4±1.06 3.20±0.37 1.2 ± 0.20 1.8 ± 1.11*
(75%
inhibition) Test(plant
extract)
Castor oil+ crude extract(200 mg/kg)
27.44±1.07 6.0 ± 0.32 1.8 ± 0.20 2.0 ± 0.84*
(72.2%
inhibition)
All values are expressed as mean±SEM (n=5). *P<0.01 significant compared to control (Students t test)
Fig 10 : Effect of ethanol extract of M. indica leaves (2 gm/kg) and commercially available antidiarrhoeal drug loperamide (1 mg/kg) on castor oil induced diarrhoea. % activities are shown within parenthess. ** P<0.01 compared with control group (Student’s‘t’ test). ↓= decrease
Discussion:
Castor oil induced diarrhoea model is widely used for the evaluation of antidiarrhoel property of drugs. Ricinoleic acid, the active metabolite of ricinoleic acid which is present in castor oil is responsible for the diarrhoea inducing property of castor oil (Gaginella and Philips, 1975). It stimulates peristaltic activity in the small intestine, leading to changes in the electrolyte permeability of the intestinal mucosa. Its action also stimulates the release of endogenous prostaglandin which stimulates motility and secretion (Pierce et al., 1971, Galvez et al., 1993). In this study, the methanolic extract of M. indica displayed a significant and dose-dependent antidiarrhoeal activity against castor oil-induced diarrhoea in mice. The results were found to be similar to that of the standard drug loperamide (1 mg/kg) with regard to the severity of diarrhea. Loperamide is widely used in the management of diarrhoeal disorders effectively antagonizes diarrhoea induced by castor oil (Niemegeers et al., 1974). The antidiarrhoeal property of the methanolic extract of M.
indica found in the present study could be owing to the presence of tannins, alkaloids, saponins, flavonoids in this plant. Previous studies showed that antidysenteric and antidiarrhoeal properties of medicinal plants were mostly due to tannins, alkaloids, saponins, flavonoids, sterol and triterpenes (Galvez et al., 1991, 1993; Longanga et al., 2000). The result obtained in this study infers the antidiarrhoeal property of the methanolic extract of M. indica.
**
75%↓ **
72.2%↓
Cytotoxicity assay of Mango( Mangifera indica)
Result of cytotoxicity effect:
In cytotoxicity study , the leaf extract of Mango( Mangifera indica) was found to be safe and no mortality was observed to a dose as high as 879.70µg/ml.
Table 5: Brine shrimp lethality bioassay of ethanol extract of Mango(Mangifera indica) leaf
Dose (µg/ml)
Log dose
Total Alive Death % (Lethality)
Actual
%
Probit
62.5 1.796 20 20 0 0 0.013 1.84
125 2.097 20 19 1 5 0.050 3.62
250 2.398 20 17 3 15 0.150 4.03
500 2.699 20 16 4 20 0.200 4.18
1000 3.000 20 14 6 30 0.300 4.49
2000 3.301 20 0 20 100 0.988 6.58
Fig 11 : Regression Line and experimental point
Table 6: Calculation of LC50 value, regression equation and confidence limit by probit analysis.
Probit analysis
Log10 LC50 LC50 (μg/ml) 95% confidence
limit (μg/ml) Regression equation Chi square
value 2.94 879.76 360.6-1365.9 Y =- 2.2776 + 2.5116 * X 6.37 Discussion:
Cytotoxic activity of Mangufera indica leaves extract was determined by Brine-Shrimp Lethality Assay. Percentage mortality of brine shrimp at 6 different concentrations (62.5, 125, 250, 500, 1000) µg/ml of Mangufera indica leaves extract is showed lethality in a dose dependent manner. More specifically, 0%, 5%, 15%, 20%, 30% and 100% mortality was observed at 62.5, 125, 250, 500, 1000 and 2000 μg/ml respectively .
From the % lethality of brine shrimp, the probits were calculated for each concentration by using computer program “BioStat-2007”. Probits were then plotted against corresponding leaves extract log concentration and from the plot LC50 (log concentration 50) value was 2.79 μg/ml calculated by regression analysis. LC50 value of Mangufera indica leaves extract was found 879.76 μg/ml with 95% confidence limit where the lower and upper limits were. 360.6,1365.9μg/ml respectively .Chi square data at 5% level for percentage of mortality of brine shrimp was 6.37.
Chapter V Conclusion
Modern scientific evaluation of medicinal plants and herbs is mainly concerned with validating the traditional use of plants and identifying the active components of extracts and preparations. While this may be important in situations where the plant in question also produces potentially toxic compounds, it is possible that a number of components could act synergistically to produce the observed therapeutic effects. Hence, separating the individual components may lead to a loss of the desired activity. As a result, continued examination of traditional plant medicines is required not only to establish the scientific basis for activity, but enable the quality, efficacy and safety of such preparations to be better assessed. Mangifera indica leaves can be considered to be very effective in preventing castor oil induced diarrhoea and possess strong antibacterial activity as well as have no cytotoxic effect in tested concentration. Further investigation warrant to isolation, identification and characterization of different active compounds from the extracts and their mode of action, responsible for these properties, on different biological systems is required.
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