Comparative Study on the Phyllanthus Acidus and Phyllanthus Embilca and their Antimicrobial Activity

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INTRODUCTION

Phyllanthus acidus, isa plant belongingto the Euphorbiaceae family. It is generallyrecognizedas the Country gooseberry. It is usuallycultivated for ornamentation and itslocalname is Arenelli (Tamil). The fruits of P. acidusare collected from different agroclimatic zones of Tamilnadu. The plant is an intermediary between trees and shrubs with edible small yellow berries fruits. Varioussignificantchemical constituents are found in this plant. The plant has medicinal usage, too. The peppered leaves are utilizedto make a poultice to treat sciatica, lumbago and rheumatism (but have been observed to cause low blood pressure when combined with nitrates), At the same time, the seeds are used as a cathartic and the root, if prepared with care, as a purgative. The syrup is used to medicate the stomach, and in India the fruit is usedas a blood-enhancer for the liver. P. acidus contains 4-hydroxybenzoic acid, kaempferol,adenosine, caffeic acid, and hypogallic acid. The medicinal activities of Phyllanthusspecies are antipyretic, analgesic, anti-inflammatory, anti-hepatotoxicand antiviral [1-5].

Phyllanthus Emblica Linn. (syn. Emblica Officinalis), usuallyknown as Indian gooseberry or amla, family Euphorbiaceae, is significant herbal drug used in Unani (Grac- Arab) and ayurvedic systems of medicine. Phyllanthus Emblica is highly nutritious and could be a significant dietary source of minerals, vitamin C, and amino acids. The plant contains emblicol, tannins, phyllembelin, phyllembelic acid, rutin, curcuminoids, and phenolic compounds. All parts of the plant are consumedfor medicinal purposes, especially the fruit, which has been used in Ayurveda as a potent Rasayana and traditional medicine to treat inflammation, jaundice, and diarrhea. Different plant parts have chemopreventive, antiulcerogenic, hypolipidemic, antioxidant, hepatoprotective, antidiabetic, gastroprotective, and antibacterialproperties. Phyllanthus Emblicais a mainingredient in many Ayurvedic preparationssuch as Chyawanprash and Triphala. The present study intended to investigate the comparative study on estimating ascorbic acid and tannins in Phyllanthus acidus and Phyllanthus Emblicaand their antimicrobial activity [6-12].

MATERIALS AND METHODS

The fruits were collected from thecentral market of Madurai city. All the informations about these two spiceswas collected from Madurai Medical College, Madurai, Al Ameencollege of pharmacy, Bangalore and American college, Madurai.

Materials used

The materialsused were soxhlet apparatus. Chloroform, methanol, and distilled water and shade dried fruits of Phyllanthus Emblica and Phyllanathus Acidus.

Purification of solvent

The various solvents Chloroform, methanol and distilled water, were purified as follows:

Chloroform

Chloroform was shaken with an equal volume of distilled water twice to remove water-soluble impurities and separated using a separating funnel. It was then dried again with anhydrous potassium carbonate for another 24 hours. It was then dried again with anhydrous potassium carbonate for another 24 hours. It was decanted and distilledthe fraction boiling high at 60°c was collected and stored in a dark brown bottle. Absolute alcohol was added as a preservative.

Methanol

Methanol was time distilled and used.

Water

Distilled water was used for water extraction.

Isolation of ascorbic acidfrom phyllanthus emblica and phyllanathus acidus

The fully ripe fruits (1 kg) were pulped and pressed and the juice (450 ml) was treated with lead acetate. 330 ml and asmall quantity of formic acid. The impurities were present in the precipitate and the ascorbic acid was present in the solution. After separation, the solution was again treated with lead acetate and ammonia was added to make it alkaline as to phenolphthalein. This timethe precipitate was isolated by centrifuging at 5000 rpm for 5 minutes. It was decomposed by adding a sufficient amount of hydrochloric acid (125ml) and the lead chloride was filtered off and washed to recover all ascorbic acid. The filtrate was concentrated rapidly under reduced pressure (150 vapor pressure at 35°c) to a syrupy liquid containing not more than20 % water. This 120 ml was extracted repeatedly with an excess of acetone (50ml). The acetone solution in which most of the ascorbic acid present was evaporated to a sticky syrup liquid 100 ml under reduced pressure (150 vapor pressure at 35°c) and again extracted with dry acetone 15 ml and then treated with butyl alcohol 20 ml and the remaining acetone was removed under reduced pressure (150 vapor pressure at 35°c) for 1 hr. The slowly crystallized from 68ml butyl alcohol solution kept at 0°c for several days. The crystals were separated usingacentrifuge at 5000 rpm for 5 minuteswashed with acetone methanol mixture, and recrystallized from a mixture of methanol and dioxane (4:1). The total amount of ascorbic acid present was found to the 25%.

Estimation of ascorbic acid usingfresh amla fruit juice

About 100mg of the powdered sample of ascorbic acid was dissolved in 100 ml of recently boiled and cooled water. Then add 25 ml of dilute sulphuric acid, and the mixture was titrated against iodine solution taken in the burette using starch mucilage as an indicator. Each ml of N/10 iodine solution was equivalent to 0.008801 gm of ascorbic acid was calculated by the formula

Isolation of tannin from phyllanthus emblica and phyllanathus acidus

The fully ripe fruits were pulped and pressed, and the juice was treated with lead acetate and some formic acid. The precipitate consists of lead tannate. Collect the precipitate by filtration. Then the precipitate was treated with hydrochloric acidlead tannate was converted into tannic acid and lead chloride. Then the lead chloride was separated by filtration. The filtration was collected and concentrated by evaporation. Finally, is left as a residue.

Antimicrobial activity

The antimicrobial activity of chloroform, and aqueous extract of Phyllanthus Emblica and Phyllanathus Acidus against the staphylococcus aureus (gram +) Escherichia coli (gram -) was carried out by cup plate method. Casein acid hydrolysate, agar, beef extract, starch were added to distilled water and dissolved by heating and filtering. The pH was adjusted to 7.6±0.2. A clear solution was obtainedand it was sterilizedby autoclaving for 175°c for 15 minutes. The media was poured into the Petri plates, and test organisms were inoculated. The temperature of the media has beenmaintained. Four holes were drilled in the media [13-15]. The two holes served as a extract for both the spices of Phyllanthus Emblica than Phyllanathus Acidus. Third hole served as a standard drug Amikacin and test used for the control. A uniform concentration of 1000µg/ml of the different extracts were found into the corresponding hole. After 24 hour of incubation of plates at 37 °c the zone of inhibition were measured in nm. The activity was compared with standard drug Amikacin and control. The antimicrobial activity is showed in (Figures 1-6).

RESULTS AND DISCUSSION

Phytochemical investigation

In Phyllanthus Emblica the phytochemical constituents present are carbohydrates, Flavonoids, Tannins, Pectin, and Ascorbic acid. In Phyllanthus Acidus, the phytochemical constituents present are carbohydrates, Tannins and Ascorbic acid.

Estimation of ascorbic acid from phyllanthus emblica and phyllanathus acidus

Phyllanthus Emblica

Percentage purity of ascorbic acid =7.3*0.008801*0.9754*100/0.0986=63.5%.

Phyllanthus Acidus

Percentage purity of ascorbic acid = 6.9*0.008806*0.9754*100/0.0975=60.78%.

Comparative Reportfor the Content of Ascorbic Acid

From the above results the content of ascorbic acid is more in Phyllanthus Emblica than in Phyllanathus.

Isolation of tannin from phyllanthus emblica and phyllanathus acidus

The amount of tannin obtained from Phyllanthus Emblica =4.1 gm

The amount of tannin obtained from Phyllanathus Acidus=2.7gm

The content of tannin is more in Phyllanthus Emblica than Phyllanathus Acidus.

Antimicrobial activity

The species of Phyllanthus Emblica and Phyllanathus Acidus have antimicrobial activity. The chloroform extract and methanolic extract show significant antimicrobial activity (Figures 1, 3, 4 and 6). The antimicrobial activity is more in Phyllanathus Acidus than in Phyllanthus Emblica (Tables 1 and 2).

Table 1. Extract of Phyllanthus Emblica and Phyllanthus Acidus against staphylococcus aureus (gram +)

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