1. INTRODUCTION:

Chronic hepatitis C infection, usually a subclinical disease in its initial phase, can be associated with progressive liver disease that may evolve insidiously into cirrhosis and carries an increased risk of hepatocellular carcinoma. (1,2) Spontaneous remission of viral replication is rare; therefore, effective treatment is highly desirable.

Treatment with α-interferon leads to serum alanine aminotransferase (ALT) normalization and HCVRNA clearance in 20–40% of patients but relapses after treatment withdrawal are frequent. (3) So different treatment strategies have to be sought for those who do not respond. These approaches might include viral activity reducing or hepatoprotective medication.

In Japan glycyrrhizin, a natural compound extracted from the roots of Glycyrrhiza glabra, has been used for more than 20 years as a treatment for chronic hepatitis. (4) Glycyrrhizin is a conjugate of one molecule of glycyrrhetinic acid with two molecules of glucuronic acid. It has been used for many centuries in traditional Chinese medicine as an anti-allergic agent. Because of its sweet taste it is also used as a food additive, for example in beverages and licorice. (5) In 1946 Revers reported the anti-ulcer effect of licorice; (6) since then, glycyrrhizin has been used for many years as an anti-ulcer drug in Europe. In Japan intravenous glycyrrhizin has been used for allergic diseases, mainly in the dermatological field. In an attempt to use glycyrrhizin as a treatment for ‘allergic’ hepatitis it was found to lower the transaminases. In 1977 Suzuki et al. performed a double-blind randomized controlled trial in 133 cases with histologically documented chronic active liver disease. (7) The plasma transaminase activity improved significantly in the group treated with glycyrrhizin compared to the placebo-treated group. Hino et al. also found an improvement in liver histology after treating chronic active hepatitis with glycyrrhizin. (8) The mechanism by which glycyrrhizin improves the biochemistry and histology is unknown. This review will deal with the metabolism and pharmacokinetics, the side-effects, and the anti-viral and hepatoprotective action of glycyrrhizin. It will also discuss the long-term effects of glycyrrhizin in the treatment of chronic viral hepatitis C.

2. MATERIALS AND METHODS:

Wood Powder were collected from local area of Bhopal Madhya Pradesh India. The voucher specimen was deposited in Department of Pharmacognosy Ajmera College of Pharmacy Nagao.

2.1 Extraction of plant material:

The collected, cleaned wood of glycyrrhizin were used for the extraction process. 200g of powder of wood were macerated with Methanol, shaking frequently during first 6 hours and allowing stand for 18 hours. The extracts were filtered through what Mann filter paper to remove any impurities if present. The extracts were concentrated by vacuum distillation to reduce the volume 1/10. The concentrated extracts were transferred to 100ml beaker and to removing solvent were evaporated on the water bath. The dried extracts were packed and labeled in air tight container for the further studies

2.2 Estimation of glycyrrhizin contents:

2.2.1 Thin layer chromatography profile of glycyrrhizin and sample preparation for Thin layer Chromatography:

Standard preparation:

Around 2ml of 99% glycyrrhizin was taken in a clean and dry test tube. The standard glycyrrhizin was spotted on the Merck aluminum plate precoated silica gel F254 with 0.2mm thickness.

Preparation of mobile phase:

95% of methanol was mixed with 5% of chloroform by ultra-sonication and taken around 100ml in a chromatographic chamber. The chamber was saturated for 30 mins to avoid edge effect.

METHOD:

After chamber saturation the spotted plate was placed in the chamber. Then the mobile phase was allow running, the present glycyrrhizin in the spot according to its affinity towards mobile phase moved and its retardation factor (Rf) was calculated.

Preparation of sample for Thin layer chromatography:

175mg of each extract was drawn and dissolved in 10ml of solvent (Meoh: Chloroform 95:5) So 1ml of solution Contains 17.5mg of extracts. This 1ml from the above solution was applied to the silica gel percolated plate (For Thin layer Chromatography) in the form of band and all the procedure was maintained as like as standard.

Chemical structure of Glycyrrhizin

2.3 UV analysis:

2.3.1 Determination of λ max:

Solvent was prepared by mixing 95 parts of methanol with 5 parts of chloroform with proper sonication. Since standard glycyrrhizin contains 99%, hence 99gm in 100ml and 1000μg contained in 1.01ml. 1.01ml dissolved up to 10ml of solvent to give 100μg/ml of glycyrrhizin. From the above 10ml contains 100μg of glycyrrhizin 1ml was drawn and again dissolved up to 10 ml of solvent to give 10μg/ml of glycyrrhizin. Again from the above 10ml contains 10μg of glycyrrhizin 1ml was drawn and dissolved up to 10 ml of solvent to give 1 μg/ml of glycyrrhizin. The prepared three concentrations (1μg/ml, 10μg/ml, 100μg/ml) of glycyrrhizin was then carried out for determination of λ max.

Standard Curve of Glycyrrhizin (UV)

Table 1. Calibration Table of Glycyrrhizin (UV)

Concentration μg/ml	Absorbance
0	0
5	0.322
10	0.623
15	0.922
20	1.272
25	1.552

2.3.2 Preparation of standard curve:

From 99% glycyrrhizin stock solution was prepared. That is 10.1ml of solution contains 99% was dissolved in 10 ml of solvent to give 1000μg. From that stock solution different Eli quotes of 1, 2, 3, 4, 8, 10, 20 and 25μg/ml was prepared and scanned in UV. The corresponding absorbance were noted. Then a calibration curve was plotted by taking Absorbance vs. Concentration.

2.4 HPLC analysis:

2.4.1 Development of method:

After all trial and error, the following developed method having 50 parts of methanol, 25 parts of water and 75 parts of acetonitrile was fit strong for glycyrrhizin in a Isocratic system and this method was developed for both standard glycyrrhizin and glycyrrhizin present in alcoholic extracts of different species of glycyrrhizin.

Table 2. Calibration Table of Glycyrrhizin (HPLC)

Concentration ng/ml	Peak Area
25	1290
50	2680
75	3976
100	5356
125	6678

Standard Curve of Glycyrrhizin (HPLC)

2.4.2 Calibration of glycyrrhizin by HPLC:

1.01ml of 99% v/v glycyrrhizin when diluted up to 10ml of solvent (MeOH: H₂O: ACN, 50:25:75) to give100μg /ml of glycyrrhizin. From this stock solution aliquots of 25, 50, 75, 100, 125 nano gram per ml of glycyrrhizin were prepared. After sufficient 30 min sonication and filtration through 0.45micron filter paper those solutions were injected to HPLC for Calibration.

3.1 Quantitative Estimation of glycyrrhizin by UV:

The concentrations of glycyrrhizin can be determined by UV-VIS spectrophotometer by putting the absorbance of glycyrrhizin obtained from various extracts of glycyrrhizin in the standard calibration curve. From the spectrum of Fig.1 it was found that the λ max of glycyrrhizin (Peak-2) is 281 nm.

Fig. 1: UV Spectrum of Standard Glycyrrhizin

Glycyrrhizin:

In Fig.2 peak of glycyrrhizin. Having absorbance 0.456 A at near about the λ max of standard glycyrrhizin. It was found from calibration curve 7.26μg of glycyrrhizin present in 1ml of Solvent. Hence 5ml of solution contains = 7.26×5=36.3 μg =0.036 mg so 1ml of Solvent which Contains 17.5mg of extract contains 0.036 mg of glycyrrhizin. Hence 5000mg (5gm) of Extract contains = 0.036/17.5×5000 mg of glycyrrhizin =10.38mg of glycyrrhizin. Hence % of glycyrrhizin = 10.38/5000 × 100 = 0.20%

Fig. 2: UV Analysis of Glycyrrhizin

3.2 Quantitative estimation of glycyrrhizin by HPLC method:

The concentrations of glycyrrhizin isolated by thin layer chromatography from alcoholic extracts of different species of glycyrrhizin can be determined by putting the peak areas of glycyrrhizin in the standard calibration curve of HPLC. The Fig.3 shows the spectrum of standard glycyrrhizin (Peak-1) at Rt 12.79.

Fig. 3: HPLC Chromatogram of Glycyrrhizin

DISCUSSION:

The result from the UV analysis it was conclude that contains 10.38mg (0.20%) of glycyrrhizin and from the HPLC analysis it was conclude that contains 8.28mg (0.16%) of glycyrrhizin from 5gm of Extract. The result Obtain from UV and HPLC analysis shows contains higher amount of glycyrrhizin and established Ultra-violet spectroscopy and high-performance liquid chromatographic method for quantification of the glycyrrhizin in the Powder of the plant has been sensitive and reliable.

REFERENCES:

1. Agrawal SS, (2007) Herbal Drug Technology, Hyderabad, India, Universities press, Vol: 1, pg. No. 8-13.

2. Ansari S.H (1998), Tata’s Guideline series of Pharmacognosy, Tata Publishers, edition Ist, 34.

3. Asolkar L.V et al (2005) Glossary of Indian medicinal plants with active principles (part 1), CSIR New Delhi; 327-328.

4. Atal C.K., Kapur B.M. (1989), Cultivation and utilization of Aromatic Plants, Reprint Edition: 1- 2, 90-92, 95, 641.

5. Brain, K. R. and Turner, T. D. (1975). The Practical Evaluation of Phytopharmaceuticals. Wright-Scientica, Bristol pp. 57-58

6. Dr. Mukherjee P.K, Quality Control Herbal Drugs 1st edition reprint 2010 published by Business Horizons New Delhi Pg. No.186-89,287-90

7. Dr. Tailang Mukul and Dr. Sharma Ashok kumar, Phytochemistry Theory and Practicals, Birla Publication Pvt. Ltd. Delhi. Pg. No. 80-83

8. Gupta S.K. et al, 2002. Validation of claim of Tulsi, Ocimum sanctum Linn as a medicinal plant, J Expt. Biol., 765-773.

Received on 25.03.2018 Modified on 13.05.2018

Res. J. Pharma. Dosage Forms and Tech.2020; 12(2): 49-52.

DOI: 10.5958/0975-4377.2020.00008.7