MEDICINAL POTENTIAL OF PHYLLANTHUS EMBLICA (LINN.) FRUITS EXTRACTS: BIOLOGICAL AND PHARMACOLOGICAL ACTIVITIESHTML Full Text
MEDICINAL POTENTIAL OF PHYLLANTHUS EMBLICA (LINN.) FRUITS EXTRACTS: BIOLOGICAL AND PHARMACOLOGICAL ACTIVITIES
S M Moazzem Hossen*, Mohi Uddin and Md. Raihan Sarkar
Department of Pharmacy, University of Chittagong, Chittagong-4331, Bangladesh
Department of Pharmacy, Jagannath University, Dhaka, Bangladesh
ABSTRACT: The objective of the present study was to investigate the scientific basis of the traditional use of the fruit of Phyllanthus emblica (Linn.). The selected pharmacological investigations are antimicrobial screening, analgesic activity, antidiarrheal activity screening and the brine shrimp lethality test for cytotoxic activity. Phytochemical analysis of ethanolic fruits extract Phyllanthus emblica (Linn.) showed the presence of flavonoids, alkaloids, tannin, steroids, reducing sugar and gum. The fruit extract produced 19.07% and 38.67% protection or writhing inhibition at the oral dose of 250 and 500 mg/kg-body weights respectively. From the observations it is obvious that the ethanol extract of the fruit of Phyllanthus emblica (Linn.) has an analgesic activity. The ethanolic fruit extract of Phyllanthus emblica (Linn.) significantly inhibited ear edema formation in xylene induced ear edema model mice. This inhibition can be considered as direct evidence that supporting the anti-inflammatory activity of ethanolic fruit extract of Phyllanthus emblica (Linn.) The ethanolic fruit extract of Phyllanthus emblica (Linn.) performing as a remedy for different bacterial diseases is supported by the antibacterial screening tests. During the antidiarrheal activity screening at the dose of 500 mg/kg-body weight, the extract of Phyllanthus emblica (Linn.) compared to the control group, offered about 0.5389 of the mean latent period for diarrheal episode to ensure and the result was significant (P<0.2). The mean numbers of stool at the 1st, 2nd, 3rd and 4th hour of study were 8.2, 9.6, 7.8 and 4.4 respectively. Phyllanthus emblica (Linn.) showed a moderate antidiarrheal activity in castor oil induced test in mice and caused an increase in latent period i.e. delayed the onset of diarrheal episode and decreased the frequency of defecation. The decreased frequency of defecation and increase mean latent period of test group than control group and comparison with positive control group can claim that Phyllanthus emblica (Linn.) might possess antidiarrheal activity. T-test of these responses showed that the result is significant throughout the observation period. The ethanolic fruits extract have cytotoxic activity and test sample showed different mortality rate at different concentrations. The LC50 values were found to be 60µg/ml for the crude extract. The 90% mortality (LC90) values were 100µg/ml respectively. The approximate significance value for Chi-Square Tests (Linear by liner association) is equal 0.02 and also significance value for Pearson’s R is 0.001 which are significant at 5% level of significant. According to the results of the present investigation, it can be concluded that the ethanolic fruits extract of Phyllanthus emblica (Linn.) has significant analgesic, anti-inflammatory, antimicrobial, anti-diarrheal and cytotoxic effects that support to the traditional use of this plant for the treatment of related diseases. This study also suggests for the further detail investigation of mechanisms of the pharmacological effects and also to isolate the active compound(s) responsible for those properties
Phyllanthus emblica (Linn.), Antimicrobial, Analgesic, Antidiarrheal, anti-Inflammatory, Bioassay.
Introduction: Phyllanthus emblica (Linn.) or Emblica officinalis (Gaertn) belongs to the Family Euphorbiaceae. Bengali/vernacular names are Amloki, Amla; Aila (Sylhet) and tribal names are Ambari (Garo); Amloti (Chakma); Soi sha (Marma); Sowan Lu (Bawm); Khulu (Murong). English names are Emblic Myrobalan, Indian Gooseberry1. Fruit a globose drupe, about 2.5 cm across, obscurely 6-lobed. Occurs in the dry forests of Chittagong, Chittagong Hill Tracts, Cox’s Bazar, Sylhet, Dhaka-Tangail (Sal forest) and Dinajpur; also cultivated elsewhere1. Fruits are diuretic, refrigerant, carminative, astringent, tonic, stomachic, laxative, antacid and rich in Vitamin C, improves appetite, useful in vomiting and burning urination, diseases of the heart and liver, piles, stops nasal haemorrhage. It promotes children’s resistance to cough and cold; used as a hair tonic. Dried fruits are useful in haemorrhoids, diarrhoea, dysentery, anemia, jaundice and dyspepsia. The fruits are also said to be beneficial in insomnia, skin problems, gall pain, leucorrhoea and tympanites. Sherbet prepared from the fruit along with lemon juice is used for arresting acute bacillary dysentery. Fruits are a valuable component of “Trifala” used in different Ayurvedic preparations. Flowers are cooling and aperient. Bark is astringent 1. Ethanolic extract of the leaves possess good antibacterial properties and mild antifungal properties 2, 3. Phyllambin isolated from fruit potentiates pharmacological action of adrenaline, has mild depressant action on central nervous system and possesses spasmolytic action 1. Water extract of the fruit causes moderate relaxation of the isolated guinea-pig ileum, but it does not interact with the activity of Acetylchloline 4.
Fruit is a rich natural source of vitamin C. It also contains tannins and colloidal substances, phyllembic acid, lipids, gallic acid, ellagic acid, trigalloylglucose, terchebin, corilagin and emblicol. Phyllembin and mucic acid have been isolated from the fruit pulp. Seeds contain fixed oil, phosphatides, tannins and essential oil. Bark, fruits and leaves are rich in tannin. They also contain lupeol, β-sitosterol and ellagic acid. Bark also contains leucodelphinidin. Seed oil also contains linoleic acid (64.8%), closely resembled linseed oil 1, 5, 6.
The selected pharmacological investigations are antimicrobial screening, evaluation of analgesic activity, antidiarrhoeal activity screening and the brine shrimp lethality test by using the ethanolic fruit extract of Phyllanthus emblica (Linn.). The present study investigates the phytoconstituents in order to correlate the folkloric claims with the bioactive compounds present in the plant and investigates the cytotoxicity potential of the plant on brine shrimp (Artemia salina) larvae. Since toxicological evaluation of plant extracts seeks to determine its possible collateral effects to ensure the safety of its use, brine shrimp larvae being sensitive to toxic substances are commonly used for toxicity assays in pharmacology 7, 8.
Material and methods:
Plant material collection and identification
Fruits of Phyllanthus emblica (Linn.) were collected from the district of Chittagong and were identified by the experts and preserved in the herbarium, department of botany, University of Chittagong. (Acc. No. SBU1521).
Plant extracts preparation
The collected fruits were separated from undesirable plant materials or plant parts. They were air-dried for one week. The fruits were ground into a coarse powder. The powder was stored in an airtight container and kept in a cool, dark and dry place until analysis commenced. Cold extraction was performed. About 350 gm of powered material was taken in a clean, flat bottomed glass container and soaked in 900 ml of 80% ethanol. The container with its contents was sealed and kept for a period of 10 days accompanying occasional shaking and stirring. The whole mixture then underwent a coarse filtration by a piece of clean, white cotton material. Then it was filtered through Whatmann filter paper [Bibby RE200, Sterilin Ltd., UK]. The filtrate (ethanol extract) obtained was evaporated under ceiling fan and in a water- bath until dried and % yield of fruit calculated 4.57% 9. The extract was stored at 4oC until use.
Young Swiss-albino mice aged 4-5 weeks, average weight 20-25 gm were used for the experiment. The mice were purchased from the Animal Research Branch of the International Centre for Diarrhoeal Disease and Research, Bangladesh (ICDDR, B). They were kept in standard environmental condition (RH 55% to 60%, room temperature 25± 2oC and 12 h light/ dark cycle) for one week for adaptation after their purchase and fed ICDDR, B formulated rodent food and water. The experimental study was performed under the guidelines of Institutional Animal Ethics Committee 10.
Chemicals and drugs
Drug Diclofenac Sodium, Kanamycin, Loperamide were from square pharmaceuticals limited and Vincristine Sulphate (VINCRIRST ®) from Techno Drugs Ltd. Used other reagents and analytical kits were laboratory reagent grade and from Merck Specialities Private Limited, India.
Preliminary phytochemical studies were performed to identify different phytochemical constituents in the extract as well as powdered drug adapting standard methods as described 11-14.
Analgesic activity of the ethanolic extract of Phyllanthus emblica (Linn.) was tested using the model of acetic acid induced writhing in mice 15-20. Young Swiss-albino mice, the experimental animals were randomly selected and divided into four groups denoted as group-I, group-II, group III and group-IV, consisting of 5 mice in each group. Group I received 1% tween 80 in water (10 ml/kg) as control, group II received standard drug Diclofenac sodium (25 mg/kg) while group III and IV received 250 mg/kg and 500 mg/kg extract of sample respectively. Each mouse was weighed properly and the dose of the test samples and control materials were adjusted accordingly. Test samples and diclofenac sodium were given orally by means of a feeding needle and the control was given intraperitoneally. After 30 minutes, the writhing inducing chemical, acetic acid solution (0.7%, 10 ml/kg) was administered intraperitoneally to each of the animals group. After an interval of five minutes the number of squirms (writhing) was counted for 15 minutes. The mice didn’t always perform full writhing. The incomplete writhing was taken as a half writhing, so two half writhing were taken as one full writhing.
Xylene induced air edema model mice were used to assess the anti-inflammatory activity of the plant extract following the method described by Dev et al. 21. Experimental mice were divided as previous test. Group I received 1% tween 80 in water (10 ml/kg) as control, group II received standard drug Diclofenac sodium (25 mg/kg) while group III and IV received 250 mg/kg and 500 mg/kg extract of sample respectively. One hour after administration of the above dose, 0.01 ml of xylene was injected to the anterior and posterior surfaces of the right ear of the each mouse. One hour after xylene injection mice were sacrificed and both treated and untreated ears were cut downed by using a 7mm diameter cork borer as circular sections and weighed. The weight difference between untreated and treated ear sections was calculated 19 - 22.
Antimicrobial screening was performed using disc-diffusion method. Sample disc 500 µg/disc and Standard Kanamycin (30 µg/disc) discs were used as positive control and blank discs were used as negative controls. The sample discs, standard antibiotic discs and control discs were placed gently on marked zones in the agar plate’s pre-inoculated with test bacteria, protozoa and fungi. The plates were then kept in a refrigerator at 40C for about 24 hours to allow sufficient diffusion of materials from discs to surrounding agar medium. The plates were then inverted and kept in an incubator at 370C for 24 hours. All organisms are listed in Table 1. Antibacterial activity of the crude ethanolic extract was determined by disc diffusion method 23 - 25. The selected organisms were Gram negative bacteria (Salmonella typi, Shigella dysenteriae, Shigella sonnei, Vibrio cholera, Hafnia, Plesiomonas), Gram positive bacteria (Staphylococcus aureus, Staphylococcus epidermis, Staphylococcus saprophyticus, Staphylococcus pyogenas) and fungi (Candida albicans, Fusarium solanii)
Young Swiss-albino mice aged 4-5 weeks, average weight 20-25gm were used for the experiment. The mice were all screened initially by giving 0.3 ml of castor oil and only those showing diarrhea were selected for the final experiment. The test animals were randomly chosen and divided into three groups having five mice in each; they were accurately weighed & properly marked of the experimental groups, group-I or the control received only distilled water containing 1% Tween-80 (10 ml/kg). Group-II or the positive control received standard anti-motility drug, Loperamide (3mg/kg) as oral suspension. The test group III was treated with suspension of fruits extract of Phyllanthus emblica (Linn.) at the oral dose of 500 mg/kg-body weight. The method, described by Chatterjee was followed for this study 26. Test samples, control and Loperamide were given orally by means of a feeding needle. The mice were fed with the samples, control and Loperamide 1 hr. prior to the oral administration of castor oil at a dose of 0.5ml per mouse. Individual animals of each group were placed in separate cages having adsorbent paper beneath and examined for the presence of diarrhea every hour in four hours study after the castor oil administration. Number of stools or any fluid material that stained the adsorbent paper were counted at each successive hour and were noted for each mouse. The latent period of each mouse also counted. At the beginning of each hour old papers were replaced for the new ones. During an observation period, the total number of faecal output including diarrheic faces excreted by the animals was recorded. A numerical score based on stool consistency was assigned as follows: normal stool=1 and watery stool=2 27 - 29.
Brine shrimp lethality bioassay
This bioassay indicates cytotoxicity as well as a wide range of pharmacological activities such as antimicrobial, anti-viral, pesticidal and anti-tumor etc. of the compounds. For cytotoxic activity of the ethanolic extracts of Phyllanthus emblica (Linn.) stock solution was prepared having a concentration of 5μg/μl. Simulated sea water was prepared by dissolving 38gm of sodium chloride per liter of distilled water and filtered. This simulated sea water was used for a hatching of brine shrimp. Test solutions of different concentration (5, 10, 20, 40, 60, 80 and 160 µg/ml) were prepared from the stock solution of plant extract and living shrimps were kept to each of the solution. After 24 hrs the test tubes were observed and the number of survived nauplii in each test tube was counted and the results were noted. From this, the percentage of lethality of brine shrimp nauplii was calculated at each concentration for each sample 30 - 34.
% mortality = × 100%
Phytochemical analysis of Phyllanthus emblica (Linn.) showed the presence of flavonoids, alkaloids, tannin, steroids, reducing sugar and gum. The results were given in Table 1.
TABLE 1: TEST RESULT FOR CHEMICAL GROUPS OF PHYLLANTHUS EMBLICA (LINN.)
EE =Ethanolic Extract, + = Present, - = Absent
Analgesic activityEffect of ethanolic fruits extract of Phyllanthus emblica (Linn.) on acetic acid induced writhing test on mice model showed mild analgesic activity.
TABLE 2: ANALGESIC ACTIVITY OF PHYLLANTHUS EMBLICA (LINN.) ON ACETIC ACID INDUCED MICE MODEL
|Animal Group and Treatment||Writhing count||%Writhing||%Writhing inhibition||(t-test)
|Group-I 1% tween-80 solution in water||36.2 ± 2.035||100||--||--|
|Group-II Diclofenac sodium 25 mg/kg||7.8 ± 1.113||21.55||78.45||(12.243)
|Group-III Extract (250 mg/kg)||29.3±
|Group-IV Extract (500 mg/kg)||22.2 ± 2.395||61.33||38.67||(4.454)
Values are expressed as mean ± SEM, SEM= Standard error of mean, n=No. of mice, %=Percentage
TABLE 3: ANTI-INFLAMMATORY EFFECT OF ETHANOLIC FRUITS EXTRACT OF PHYLLANTHUS EMBLICA (LINN.) ON XYLENE INDUCED EAR EDEMA MODEL.
|Group||n||Increased weight||Inhibition rate %|
|Blank (Xylene 0.01mL. injection)||5||10.5±0.22||00|
|Positive control (Diclofenac sodium 10mg/Kg)||5||6.5±0.23*||35|
|Test 1, Extract (250 mg/kg)||5||7.25±0.25*||18|
|Test 2, Extract (500 mg/kg)||5||6.75±0.22*||32.5|
* P<0.01, vs. blank control group.
The fruits extract of Phyllanthus emblica (Linn.) showed mild antimicrobial activity and may be a remedy for different bacterial diseases is supported by the antibacterial and antifungal screening tests.
TABLE 4: IN VITRO ANTIMICROBIAL ACTIVITY OF ETHANOLIC FRUIT EXTRACT OF PHYLLANTHUS EMBLICA (LINN.) BY FOLLOWING DISC DIFFUSION METHOD.
|Diameter of zone of inhibition in mm|
|Bacterial Gram negative|
|Bacterial Gram positive|
R= Resistant or No growth.
The fruit of Phyllanthus emblica (Linn.) performing as a remedy for different bacterial diseases is supported by the antibacterial screening tests.
Phyllanthus emblica (Linn.) showed a moderate antidiarrheal activity in castor oil induced test in mice model. Antidiarrheal activity results are summarized in Table 5 and Figure 1.
TABLE 5: EFFECT OF PHYLLANTHUS EMBLICA (LINN.) ON CASTOR OIL INDUCED DIARRHEA IN MICE
0.399 ± 0.0396
0.929 ± 0.0475
|Extract of Phyllanthus emblica Linn. 500mg/kg||E1||
0.497 ± 0.0419
Values are t-test. (n=5), cp< 0.2, a p< 0.001, b p< 0.01 vs. Control. Student’s t-test.
FIG. 1: EFFECT OF LOPERAMIDE AND PHYLLANTHUS EMBLICA (LINN.) ON CASTOR OIL INDUCED DIARRHOEA IN MICE THROUGH THE OBSERVATION PERIOD (4HR).
Test sample showed different mortality rate at different concentrations. The mortality rate of brine shrimp increased with the increase in concentration of the sample and plot of percent mortality versus log concentration on the graph paper produced an approximate linear correlation between them. From the graph (Figure 2) the concentrations at which 50% mortality (LC50) of brine shrimp nauplii occurred were obtained by extrapolation. The values were found to be 60µg/ml for the crude extract. The 90% mortality (LC90) values were 100µg/ml respectively.
TABLE 6A: RESULT OF BRINE SHRIMP LETHALITY BIOASSAY OF ETHANOLIC EXTRACT OF FRUIT OF PHYLLANTHUS EMBLICA (LINN.)
|No. of alive shrimp||% mortality||LC50
Ethanolic fruits extract of Phyllanthus emblica (Linn.)
TABLE 6B: TEST SIGNIFICANCE ANALYSIS OF BRINE SHRIMP LETHALITY BIOASSAY OF ETHANOLIC EXTRACT OF FRUIT OF PHYLLANTHUS EMBLICA (LINN.)
|Ethanolic Extract of Phyllanthus emblica (Linn.)|
|Name of applied test||Chi-value||Sig.|
FIGURE 2: LC50 AND LC90 OF PHYLLANTHUS EMBLICA (LINN.)
Discussion: Phytochemical analysis of Phyllanthus emblica (Linn.) showed the presence of flavonoids, alkaloids, tannin, steroids, reducing sugar and gum. Further studies are suggested to isolate its active components by using bioactivity guided approach.
The ethanol extract of the fruit of Phyllanthus emblica (Linn.) was subjected to acetic acid induced writhing method in mice for preliminary analgesic activity screening. The results of analgesic activity screening (Table 2) showed that the ethanol extract might possess analgesic activity depending upon the nature of their active ingredients in the extracts. The fruit extract produced 19.07% and 38.67% protection or writhing inhibition at the oral dose of 250 and 500 mg/kg-body weights respectively. Acetic acid causes pain and localized inflammation by the action of prostaglandins production [mainly, prostacyclines and prostaglandin-E (PG-E)] which have been reported to stimulate the Aδ-fibres that cause a sensation of sharp well localized pain 34. It has also been reported that acetic acid induces the increased level of PGE2 and PGF2α in the peritoneal fluid which is responsible for pain production 35-40. There is various peripherally acting analgesic drugs such as ibuprofen, aspirin, diclofenac sodium and indomethacin that have been reported to inhibit acid induced writhing by inhibition of prostaglandin synthesis 41. Therefore it can be concluded that any agent that reduces the number of writhing will demonstrate analgesic effect by inhibition of prostaglandin synthesis, a peripheral mechanism of pain inhibition 42. The result of the plant extract in acetic acid-induced writhing method suggests that the reduction of pain might be occurred due to the presence of analgesic properties in the extract via inhibition of prostaglandin synthesis. However further study should be done for its isolated, purified active components. From the above observations it is obvious that the ethanol extract of the fruit of Phyllanthus emblica (Linn.) has an analgesic activity.
Xylene is known to cause severe vasodilation and edematous changes of skin as signs of acute inflammation 43. The increased thickness of the ear tissues is caused by these histopathological changes. In the present investigation; the plant extract significantly inhibited the xylene-induced increases in ear weight in a dose related manner. This inhibition capacity of the plant extract can be regarded as the evidence of anti-inflammatory efficacy through reducing vasodilation and so that improving edematous condition. In this study the ethanolic fruit extract of Phyllanthus emblica (Linn.) significantly inhibited ear edema formation in xylene induced ear edema model mice. This inhibition can be considered as direct evidence that supporting the anti-inflammatory activity of ethanolic fruit extract of Phyllanthus emblica (Linn.)
The ethanolic fruit extract of Phyllanthus emblica (Linn.) performing as a remedy for different bacterial diseases is supported by the antibacterial screening tests.
During the antidiarrhoeal activity screening at the dose of 500 mg/kg-body weight, the extract of Phyllanthus emblica (Linn.) compared to the control group, offered about 0.5389 of the mean latent period for diarrhoeal episode to ensure and the result was significant (P<0.2). The mean numbers of stool at the 1st, 2nd, 3rd and 4th hour of study were 8.2., 9.6, 7.8 and 4.4 respectively.
Phyllanthus emblica (Linn.) showed a moderate antidiarrhoeal activity in castor oil induced test in mice at the dose of 500 mg/kg-body weight as compared to the standard antidiarrhoeal agent loperamide. Phyllanthus emblica (Linn.) caused an increase in latent period i.e. delayed the onset of diarrhoeal episode and decreased the frequency of defecation. The decreased frequency of defecation and increase mean latent period of test group than control group and comparison with positive control group can claim that Phyllanthus emblica (Linn.) might possess antidiarrheal activity. T-test of these responses showed that the result is significant throughout the observation period.
Test sample showed different mortality rate at different concentrations. The mortality rate of brine shrimp was found to be increased with the increase in concentration of the sample and plot of percent mortality versus log concentration on the graph paper produced an approximate linear correlation between them. From the graph (figure) the concentrations at which 50% mortality (LC50) of brine shrimp nauplii occurred were obtained by extrapolation. The values were found to be 60µg/ml for the crude extract. The 90% mortality (LC90) values were 100µg/ml respectively. The approximate significance value for Chi-Square Tests (Linear by liner association) is equal 0.02 and also significance value for Pearson’s R is 0.001 which are significant at 5% level of significance. Therefore mortality rate of shrimp due to Ethanolic extract concentration is not due to chance. So we can conclude there is a statistically significant relationship between Ethanolic extract concentration and mortality rate of shrimp. The crude extracts Phyllanthus emblica (Linn.) were found to show high lethality against the brine shrimp nauplii. These results tend to suggest its possible antitumor, antibacterial or pesticidal activities. However, further researches are necessary particularly with its purified fraction.
The preliminary phytochemical analysis of the plant extract showed the presence of reducing sugars, alkaloids, flavonoids, tannins, steroids, gums and glycosides. The previous scientific studies have been reported that alkaloids, flavonoids and tannins are known to inhibit prostaglandin synthetase that is responsible for its antinociceptive and anti-inflammatory effects 20, 44-50. Therefore antinociceptive and anti-inflammatory effect of the extract may be due to the presence of flavonoids, tannins, and alkaloids either singly or in combination. Presence of tannins, alkaloids, flavonoids, sterol and reducing sugars in the medicinal plants have also been known to indicate antidairrheal activity 51-52. In general antidiarrhoeal activity of tannins and flavonoids has been recognized for the inhibition of intestinal motility, antimicrobial action and antisecretory effects 48. In addition, the astringent properties of tannins are known to cause antinociceptive, antilammatory and antidiarrheal effects 20,44,53. Therefore antinociceptive and anti-inflammatory effect of the extract may be due to the presence of flavonoids, tannins, and alkaloid either singly or in combination. Besides alkaloids, flavonoids or tannins may also be responsible for anti-diarrheal potential of the plant extract.
Conclusion: According to the results of the present investigation, it can be concluded that the ethanolic fruits extract of Phyllanthus emblica (Linn) has significant analgesic, anti-inflammatory and anti-diarrheal effects that support to the traditional use of this plant for the treatment of related diseases. This study also suggests for the further detail investigation of mechanisms of the pharmacological effects and also to isolate the active compound(s) responsible for those properties.
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How to cite this article:
Hossen SMM*, Uddin M and Sarkar R. Md: Medicinal potential of Phyllanthus Emblica (linn.) Fruits extracts: biological and pharmacological activities. Int J Pharmacognosy 2014; 1(5) 307-316: .doi: 10.13040/IJP. 0975-8232.1(5). 307-316.
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S M Moazzem Hossen*, Mohi Uddin and Md. Raihan Sarkar
Department of Pharmacy, University of Chittagong, Chittagong-4331, Bangladesh
19 March, 2014
22 April, 2014
28 April, 2014
01 May, 2014