Collection and Preparation of Sample The leaves of Psidium guajava was collected from Noakhali Science and Technology University, Bangladesh. The leaves were washed properly and then air dried for several days. The leaves were then ground into coarse powder using high capacity grinding machine.
Proximate Analysis Proximate analysis of a substance constitutes different classes of nutrients present in the samples such as moisture, total ash, acid insoluble ash, water soluble ash content and extractive values.
Determination of moisture content Accurately weighed 5 g of powdered of Psidium guajava leaves were taken in a crucible. It was kept in a hot air oven at 105 – 110 0C, until free from moisture. The percentage of moisture content was then calculated with reference to the air-dried sample.
Determination of total ash value Accurately weighed 5 g of powdered Psidium guajava leaves were taken in a dried silica crucible. It was incinerated at 450 0C temperature, until free from carbon and then cooled. The weight of ash was taken and the percentage of it was calculated with reference to the airdried sample.
Determination of acid insoluble ash value The total ash obtained was boiled for 5 minutes with 25 ml of 2 N HCl, filtered and the insoluble matter was collected on ashless filter paper. Then, it was washed with hot water, ignited in silica crucible for 15 minutes at temperature not exceeding 450 0C, cooled and weighed the obtained residue. The percentage of acid insoluble ash was calculated with reference to the air-dried sample.
Determination of water soluble ash value The total ash obtained was boiled with 25 ml of water for few minutes, filtered and the insoluble matter was collected on ashless filter paper. Then, it was washed with hot water, ignited in silica crucible for 15 minutes at temperature not exceeding 450 0C, cooled and weighed the obtained residue. The difference in weight represents the water soluble ash. Finally, the percentage of water soluble ash was calculated with reference to the air-dried sample.
Determination of petroleum ether, chloroform, methanol and water-soluble extractive value 20 g of air dried, coarsely powdered Psidium guajava leaves were macerated with 100 ml of petroleum ether in a closed flask for 24 hrs, shaking frequently during the first 6 hrs and was allowed to stand for 18 hrs. Then it was filtered rapidly and precautions were taken against loss of petroleum ether. 25 ml of the filtrate was evaporated to dryness in a Petri dish, dried at 105 °C and weighed. The percentages of petroleum ether soluble extracts were calculated with reference to the air dried sample. The procedure followed as above using chloroform, methanol and water instead of petroleum ether.
Sequential Extraction The method is based on the extraction of active constituents present in the drug using various solvents ranging from non-polar to polar. The solvents used are petroleum ether, chloroform, methanol and water. The successive solvent extraction procedure was adopted for the preparation of various extracts of Psidium guajava leaves. The materials were subjected to successive extraction with solvents in their ascending order of polarity (non-polar to polar). In this process, the substance which is soluble in a solvent with particular range of polarity was extracted in the solvent and remaining marc further extracted with next solvent. The powder (200 g) was extracted sequentially for 8 hours in petroleum ether, chloroform and methanol using a Soxhlet apparatus. After methanol extraction, the remaining dried marc was extracted with water to get water extract. For the preparation of aqueous extract, the above dried marc was macerated for 3 days with distilled water and the residue was removed by filtration and filtrate was concentrated to obtain aqueous extract. All the extracts were concentrated with a rotary evaporator and dried using oven dryer at 35-40 0C. Dried extracts were stored for further use.
Test for alkaloids Solvent free extracts, 50 mg was stirred with few ml of dilute hydrochloric acid and filtered separately. The filtrate was tested carefully with various alkaloid reagents as follows: Mayer’s test: To 1 ml filtrate of the extract, 0.5 ml of Mayer’s reagent (potassium mercuric iodide) was added by the side of the test tube. A white or creamy or yellow colour precipitate was formed and that was indicated as the presence of alkaloids. Wagner’s test: To 1 ml filtrate of the extract, 0.5 ml of Wagner’s reagent was added by the side of the test tube. Reddish brown precipitate was formed and that was indicated as the presence of alkaloids. Hager’s test: To 1 ml filtrate of the extract, 0.5 ml of saturated picric acid solution (Hager’s reagent) was added by the side of the test tube. Yellowish precipitate was formed and that was indicated as the presence of alkaloids. Dragendroff’s test: To 1 ml filtrate of the extract, 0.5 ml of Dragendroff’s reagent (bismuth nitrate) was added by the side of the test tube. A prominent yellow or orange brown precipitate was formed and that was indicated as the presence of alkaloids.