Materials

Licorice was purchased from the local market and authenticated from Pharmacopoeial Laboratory of Indian Medicine (PLIM); Standard glycyrrhizic acid was procured from sigma Aldrich. All the solvents used were of analytical grade.

Physicochemical analysis

Total ash: Total ash was calculated by incinerating the fine powder of crude drug (2g) in a tarred silica crucible at the temperature of 450ºC such that there is complete removal of carbon. After that the ash obtained was allowed to cooled and weighed. The percentage of total ash was calculated using the weighed value of ash and powdered crude drug.

Acid insoluble ash: The ash value was determined for detecting the undesirable or harmful or earthy matter which can be present in the crude drug. For estimating acid insoluble value, the ash obtained from the above method was poured in 25 ml of dil. HCl kept on heating mantle. The mixture was filtered using the ash less filter paper, washed with hot water, ignited and weighed.

Water soluble ash: For determining the water soluble ash value the ash obtained from the total ash procedure was used and mixed with 25 ml of water. The mixture was filtered and the mixture obtained on the filter paper was collected and weighed. This weighed quantity of insoluble matter was subtracted from the weighed of ash for obtaining water soluble ash value. This weighed quantity was used for calculating the percentage of water soluble ash value.

Determination of Extractive values

Alcohol and water extractive values were determined using the same procedure except the use of alcohol to determine alcohol extractive value and the use of water in case of calculating water soluble extractive value. To determine the extractive value, powdered crude drug was macerated with the respective solution (alcohol for alcohol extractive value and water for water extractive value) in a closed flask for 24h (shaking frequently for the starting six hours). The solution was filtered and 25 ml of the filtrate was evaporated to dryness in tarred petri plate. The solution was kept at 105ºC and the finally the rest amount was weighed. ⁹ , ¹¹

Extraction of glycyrrhizic acid from licorice root

GA was extracted by using the method of maceration with slight modification in the method described in literature. For this purpose, drug (licorice roots) powders was macerated with the solvent mixture of acetone and dilute nitric acid for 2 h. The contents were filtered and additional 20 ml of acetone was added to the marc and warmed gently. The contents were filtered and filtrate was obtained. To this filtrate sufficient volume of dilute ammonia solution was added till precipitation of ammonium glycyrrhizinate is completed. The precipitate was collected and washed with 5 ml of acetone, dried and collected ¹² .

Phytochemical screening of GA

Phytochemical screening of GA was performed for the identification of phytoconsituents present.

Test for saponins (foam test)

The extract was dissolved with 20 ml of distilled water and stirred for 15 minutes. The formation of 1cm layer of foam for a period of time showed the presence of saponins.

Tests for flavonoids

With sodium hydroxide: Extract was mixed with 1 ml of sodium hydroxide solution. Blue to violet color indicates the presence of anthocyanins, yellow to orange color shows the presence of flavonones and yellow color indicates flavones.

With concentrated sulphuric acid: Extract was mixed with concentrated sulphuric acid. Yellow orange color indicates the presence of anthocyanins, orange to red color indicates the presence of flavones.

Shinoda test: For performing shinoda’s test, extract was dissolved in ethanol, to which magnesium turnings were added. To this mixture, Conc. Hydrochloric acid was added. Turning of magenta to purple color indicates the presence of flavonoids.

Test for terpinoids

Lieberman’s test: Acetic acid was added to the extract kept on the hot plate. To this mixture, concentrated sulphuric acid was added. Presence of pink color indicates the presence of triterpinoids in the extract.

Trichloroacetic acid test: tricholoroacetic aid was added to the extract. Formation of yellow color indicates the presence of terpinoids.

Fehling’s test

On the water bath, Extract was kept. To which Fehling solution A and B were mixed. Brick red precipitate showed the presence of reducing sugars. ⁹ , ¹³ , ¹⁴

Loss on drying

Loss on drying was calculating by the mentioned procedure. Weighed quantity of extract was poured onto a weighed petri plate. The petri plate was kept in oven and weighed at different time interval at 105ºC, till two consecutive weighing didn’t differ by more than 0.25mg which indicates the final loss of moisture present in the drug. Percentage loss on drying was calculated using the below mention formula ⁹ , ¹⁵ , ¹⁶ .

LOD(%) = weight of porcelain dish with drug at time 0 – weight of porcelain dish after 6 h ÷ weight of porcelain dish at time 0 – weight of empty porcelain dish

pH determination

The extract was dissolve in 10 ml of distilled water for evaluating the pH. The pH was determined using digital pH meter. The pH was measured in triplicate.

Melting point range

The melting point was determined by capillary technique. The drug was filled in the capillary tube, sealed to the one end. The capillary was introduced into digital melting point apparatus. The temperature, at which the drug melts, signifies the melting point of drug.

UV spectral analysis

A UV spectrum of the extracted GA was obtained by scanning the extract solution in the range of 200-800nm using UV spectrophotometer. For this, stock solution of 100µg/ml of GA solution was prepared ¹⁷ .

Analysis using Thin Layer Chromatography (TLC)

For TLC test solution, standard solution and developing solvent system were prepared. For preparing test solution, alcohol and water (7:3) was mixed to licorice extract. The solution was heated using water bath for 5 minutes, cooled and filtered. Standard solution was prepared by dissolving 5 gm of standard glycyrrhizic acid in 1ml of a mixture of alcohol and water (7:3). Developing solvent system contains the mixture of butyl alcohol, water and glacial acetic acid (7:2:1). TLC plates were prepared using silica gel solution and the retention value (Rf.) was calculated. The plates were kept in developing solvent system and examined under UV light at 254nm ¹⁸ .

In vitro anti inflammatory activity

The In Vitro anti inflammatory activity of GA was evaluated by albumin denaturation technique as given by Mizushima and kobayashi with slight modification. Extract was mixed with 1% aqueous solution of fetal bovine albumin. pH of the mixture was adjusted using 0.1NHcl. The solution was kept in a incubator at 37ºC for 20 min ¹⁹ , ²⁰ , ²¹ . Afterwards, denaturation was induced by keeping the reaction mixture at 60±1°C in water bath for 10 min. The mixture was cooled and the turbidity was measured using UV spectrophotometer. Percentage inhibition was calculated using the following equation. For this, acetyl salicylic acid was considered as a standard and the solution containing no drug was considered as control ²² , ²³ , ²⁴ .

% Inhibition = (Absorbance of control – Absorbance of sample) × 100 / Absorbance of control

Physicochemical analysis

Physicochemical parameters like ash value, acid insoluble, water soluble and alcohol soluble value were carried out. The results showed that all the values obtained were in the limit as given in Ayurvedic Pharmacopoeia of India (API). The total ash value was found to be 3.75%. The values of acid insoluble, water soluble extractive and alcohol soluble extractive value were found to be 1.93%, 3.51% and 2.18% respectively as given in Table 1 , indicating the presence of water soluble components than alcohol soluble components.

Phytochemical screening of GA

The tests performed for Phytochemical screening showed the presence of flavonoids, saponins and triterpinoids in the extract (GA) as shown in Table 2.

The extract was evaluated for the parameters like pH, melting point and loss on drying. The loss on drying after 6 hours was found to be 6.5%. According to the monograph, the extract should not loose not more than 12.0% of its weight. The value for loss on drying has been summarized in Table 3. The pH of the extract was evaluated in triplicate and the average was found to be 5.53 ± 0.05 as depicted in Table 4. The melting point was found to be -240ºC, which is comparable to the reported melting point of GA i.e.-220ºC.

UV spectral analysis

Spectral analysis was evaluated using UV spectrophotometer. The highest peak was attained at the wavelength of 230nm. As reported, the standard wavelength of GA is 254nm. The graph obtained via UV spectrophotometer has been depicted in Figure 1.

Figure 1. UV spectra of Licorice root Extract showing peak at 230nm. 

Thin layer chromatographic analysis

Rf value was obtained through TLC analysis by dividing the distance travelled by solute from the distance travelled by solvent. The Rf value was found to be 0.5 cm. The TLC plate showed purple color when seen through UV florescent light.

Rf = Distance travelled by solute / Distance travelled by solvent

= 3.5 / 7.0

= 0.5 cm

In vitro Anti-inflammatory activity

In vitro anti inflammatory studies were carried to relate anti inflammatory activity of GA with standard drug, salicylic acid (most commonly used as anti inflammatory agent). Percentage inhibition was calculated using the absorbance and it was found that the extract and standard showed 27.11 % inhibition and 49.15 % inhibition respectively. The results have been compiled in Table 5 .