Background: The present study is an attempt to evaluate and standardize three samples of Glycyrrhiza glabra roots from different geographical areas.
Material and methods: The preliminary physico-chemical, qualitative, quantitative phytochemical investigation of the roots of G. glabra from different locations was done as per the procedures specified in standard literature. The three samples were also subjected to chromatographic fingerprinting by HPTLC and HPLC using glycyrrhizin ammonical hydrate as standard. The dried roots were extracted successively with various solvents with increasing polarity and all the extracts were subjected to phytochemical screening for the identification of phytoconstituents.
Results: The presence of carbohydrates, proteins & amino acids, glycosides, saponins, phytosterols, fixed oils, gums & mucilage, flavonoids and resins was revealed by qualitative examination of the various extracts of G. glabra roots. The quantitative analyses further substantiated the findings that saponins are present in significant amounts and alkaloids are absent in the drug. The chromatographic fingerprinting data conformed to the bands and peaks in HPTLC and HPLC analyses respectively in comparison to the glycyrrhizin ammonical hydrate values.
Conclusion: The results in this study can be used for the genuine identification of the plant from its adulterants and subsequent screening for potent bioactivity.

INTRODUCTION

Quality evaluation is a fundamental requirement of industry and other organizations dealing with local traditional medicines and herbal products. The growing use of botanicals (drug and other products derived from plants) by the large populations is forcing the need for new methods to assess the health claims of these agents and to develop standards of quality and manufacture (Choudhary and Sekhon, 2011). A major problem in this regard is the presence of substitute or counterfeit herbal materials often found in the market. Even for the right species, the chemical composition and concentrations of bioactive compounds may vary dramatically with different collection seasons and regions as well as storage. Thus, it becomes necessary to chemically standardize the herbal extracts or products for biological, pharmacological and clinical studies (Liu, 2011). It has also been observed that individual species show highly varying chemical constituents (Nomura and Fukai, 1998). Several pharmacopoeias require definite species, such as G. glabra L. by the European Pharmacopoeia (European Pharmacopoeia, 2013).
G. glabra is a native of the Mediterranean and distributed in the sub-tropical and warm temperatures of the world. It is a tall, up to 2m high perennial herb found cultivated in Europe, Persia and Afghanistan and to a little extent in some parts of India (Ross, 2003; The Ayurvedic pharmacopeia of India, 2007).
It is traditionally used in China, Morocco, South Korea, Thailand, Turkey and USA. In India it is very much extensively used in Indian systems of medicines (Ross, 2003). The drug also possesses a wide reputation in the works of Arabic and Persian physicians (Singh et al., 2006). The guidance of ethnopharmacological knowledge in studying natural products has significantly contributed to the discovery of novel chemical structures and mechanisms of action (Liu, 2011). In Ayurveda the roots are alexiphermic alterant, aphrodisiac, demulcent, diuretic, useful in bronchitis, cough, fever, hoarseness of voice, gastric troubles, and urinary diseases; root- decoction is a good wash for greying and falling hair (Bakshi et al., 2001).
Continuous collection of the crude drugs by persons without knowledge led to the irrational harvesting of crude drugs. Due to this ecological imbalance and extinction of crude drugs, it paved way for substitution by sub-standard debased drugs which resulted in high cost of such drugs (Tewari, 2000).
There are references stating that particular drugs can be substituted by other Ayurvedic drugs with similar Dravyas (properties) (Vaishy et al., 1981).
With the increase in knowledge about the bioactive and main compounds in most of the commonly used herbs and the popular application of various analytical instruments such as HPLC, equipped with UV, MS and other detectors, fingerprint chromatograms are becoming more powerful qualitative and quantitative methods for standardization of herbal medicines (Liu, 2011).

MATERIAL AND METHODS

Plant Material
Two drug samples of the roots of Glycyrrhiza glabra were purchased from the local market in Bangalore, Karnataka and one sample from Nahan, Himachal Pradesh. The morphological identification of the drugs was done by comparison with published literature. The drugs were authenticated by qualified plant taxonomists. Voucher specimens were deposited at Raw Drug Collection center, FRLHT, Bangalore.
Proximate analysis
The moisture content, total ash value & acid insoluble ash value, alcohol soluble extractive values and water soluble extractive values were determined.
The moisture content was determined using infrared moisture balance (Model-M-3A Deluxe Voltag-230VAC) was used for the determination of moisture content. Respective moisture content (%) for all the samples were calculated (Manual of infrared moisture balance, Model M.3A Deluxe).
The total ash value and the acid insoluble ash were calculated as per the procedure mentioned in the Ayurvedic Pharmacopoeia of India (WHO, 1998).
The determination of water and alcohol soluble extractive value is used as a means of evaluating the quality and purity of drugs whose constituents cannot be readily estimated by other means (Liu, 2011). The determinations were carried out as per the procedure in The Ayurvedic Pharmacopoeia of India, 2004.
Calculation
The Percentage of Alcohol/Water soluble extractive value = B - A X 4 X 100 / W
where, A-Empty weight of the dish (g)
B-Weight of dish + residue (g)
W-Weight
Preparation of the G. glabra root extracts
The powdered drug (100 g) was extracted successively using a soxhlet extractor with each 200 ml of n-hexane, benzene, chloroform, ethyl acetate, acetone, ethanol and water. The extracts were filtered followed by concentrating and complete solvent evaporation. Then each of these solvent extracts was weighed and preserved at 5°C in an airtight bottle until further use (Table 2). These extracts were subjected to phytochemical screening.
Phytochemical screening
Preliminary phytochemical analysis of n-hexane, benzene, chloroform, ethyl acetate, acetone, ethanol and water extracts was carried out for the identification of carbohydrates, proteins & amino acids, glycosides, alkaloids, saponins, polyphenols, phytosterols, fixed oils, gums and mucilages, flavonoids and resins (Trease and Evans, 2009).
Quantitation
Determination of the total alkaloidal content
Alkaloids are weak bases. Alkaloids in the plant material are extracted based on their solubilities. Alkaloidal bases are soluble in non-polar solvents like chloroform and ether while their salts are soluble in water. Determination of the total alkaloidal content was done as per the procedure described in USP.
Determination of total saponins
Saponins are characterized by their haemolytic activity and foaming properties. The presence of both polar (sugar) and non-polar (steroid or triterpene) groups provides saponins with strong surface-active properties (Harinder et al., 2007).
The method followed was as per reported in Rajpal, 2002.

Calculation
% of saponins = B - C / A x 100
Where, A = the sample weight in g. B = the weight of tarred dish + saponins.
C = the weight of tarred dish.

Thin Layer Chromatography
Preparation of extracts
Powdered sample (1 g) was refluxed with 50 ml 70% ethanol for 30 min. The solution was filtered and again the marc was extracted with the same menstrum until colourless. The extracts were pooled and evaporated on a water bath and the residue was redissolved in the same solvent and made up to 50 ml. Freshly prepared extracts were used for TLC studies (Rajpal, 2002).
Preparation of standards
Standard Glycyrrhizin ammonical hydrate (NLT 91.7%) purchased from Natural Remedies Pvt. Ltd., Bangalore was prepared by dissolving 5 mg of the standard in 5 ml of HPLC water (5mg/ml).
Thin Layer Chromatographic (TLC) analysis of extracts
TLC profiling of 70% ethanolic extracts of the three samples of G. glabra was done for the identification of similar phytoconstituents between the two drugs. The solvent system Chloroform: methanol: glacial acetic acid: water (8: 4: 1.5: 1) designated as solvent system A was standardised for the drug with respect to the standard used.
High Performance Thin Layer Chromatography (HPTLC) fingerprinting
TLC procedure was standardised with the solvent system A which was used for the HPTLC fingerprinting of the three samples of the G. glabra roots along with the respective standard.
The solvent system A used has been reported for standard Glycyrrhizin ammonical hydrate.
HPTLC fingerprinting analysis was done using the CAMAG linomat 5 sample applicator using micro syringe (100µl, Hamilton), CAMAG reprostar 3,Twin trough chamber, Dip tank, Win cat software- Version 1.3.3., with a development distance of 70 mm. Pre-coated silica gel plate 60F254 from Merck (20X10cm). The solvent system optimized was Chloroform: methanol: glacial acetic acid: water (8: 4: 1.5: 1). Samples and standards were applied as bands on the plates in volumes 10 µl and detection was done under UV 254, UV 366 and derivatization with anisaldehyde sulphuric acid reagent.

Table 1. Pattern of sample & standard application for HPTLC analysis. Click here to view full image

High Performance Liquid Chromatography (HPLC)
The aqueous extracts of G. glabra roots were subjected to HPLC analysis for the observation of similar peaks between the drug samples and the respective standard glycyrrhizin ammonical hydrate.
Preparation of extracts
Sample of G. glabra roots weighing 0.125 g pulverized (16 mesh size) was extracted using water (25 ml) as solvent and sonicated for 30 min. The extract was filtered and made up to 25ml with the same solvent and filtered through 0.45 um membrane filter and used for HPLC analysis (Rauchensteiner , 2005).
HPLC method
System : Shimadzu HPLC system
Pump : LC-10ATVP pump
Injector : rheodyne injector
Detector : SPD 10AVP UV-Visible detector
Data integration : CLASS-VP6 software
Stationary phase : Merck C18 (250 x 4.6mm) column with
5µ particle size
Injection volume : 20 µl
Flow rate : 1.5ml/min
Equilibration time: 1 hr
Run time : 45 min
Detection : UV 254 nm
Elution type : Gradient
Solvent system: A- Potassium dihydrogen orthophosphate
(KH2PO4) Buffer
B- Acetonitrile
Time programme

The method described above was used for the HPLC analysis of the two drug samples along with the respective standards. Click here to view full image

RESULTS

The morphology of the G. glabra roots was in accordance with the reported literature (The Ayurvedic pharmacopeia of India, 2007). Voucher specimens deposited were designated as L/05/04/019 (Bangalore, Karnataka, India), L/05/05/027 (Bangalore, Karnataka, India) and L/07/02/012 (Nahan, Himachal Pradesh, India) for Glycyrrhiza glabra (Fig. 1).
Proximate analysis
The Moisture content, total ash, acid insoluble ash, water and alcohol soluble extractive values for G. glabra were determined (Table 1). All the parameters were found to be within the prescribed limits (The Ayurvedic pharmacopeia of India, 2007).
Extracts
In our study the yields (% w/w of the powdered drug) of seven extracts viz n-hexane, benzene, chloroform, ethyl acetate, acetone, ethanol and aqueous extracts were found to be 400 mg (0.40% w/w), 700 mg (0.70% w/w), 500 mg (0.50% w/w), 830 mg (0.83% w/w), 13.56 gm (13.56% w/w), 13.22 gm (13.22% w/w) and 17.82 gm (17.82% w/w) respectively. The maximum yield was observed for the aqueous extract.
Phytochemical screening
All the seven extracts were screened for phytoconstituents as shown in Table 4. The ethyl acetate, ethanol and water extracts showed the presence of carbohydrates. Proteins & amino acids were found to be present only in the aqueous extract. It was notable to observe the presence of saponins and gums & mucilages in the alcoholic and aqueous extracts. The benzene, ethyl acetate, acetone, ethanol and water extracts showed the presence of fixed oils & fats. The flavonoids were present in the acetone, ethanol and aqueous extracts. Alkaloids and polyphenols were found to be absent in all the extracts and glycosides were present in the benzene extract alone. Phytosterols were identified in the n-hexane, ethyl acetate, acetone and alcoholic extracts. Resins were found to be present in the non-polar solvents i.e., n-hexane, benzene and chloroform extracts.
Quantification
The quantitative analyses supported our phytochemical screening that alkaloids are absent in the drug. The total
saponins in the methanolic extract were found to 12.48% w/w. However, further study is necessary to isolate and quantify active constituents present in the roots of G. glabra by sophisticated techniques.

Table 2. Determination of Proximate Analysis of Glycyrrhiza glabra. Click here to view full image

Table 3. Phytochemical composition of the extracts of Glycyrrhiza glabra roots Click here to view full image

Thin Layer Chromatography
TLC finger printing of Glycyrrhiza glabra roots was done along with standard glycyrrhizin ammonical hydrate under UV 254, UV 366 nm and after derivatisation with anisaldehyde sulphuric acid reagent. A solvent system Chloroform: methanol: glacial acetic acid: water (8: 4: 1.5:1) was optimized with respect to the standard used which gave a good separation and resolution of constituents with maximum number of bands.
High Performance Thin Layer Chromatography (HPTLC) fingerprinting
The HPTLC chromatogram of the plates developed with the solvent system A are shown in figures 2, 3 and 4. The results are tabulated in Table 4.

Figure 1-3. HPTLC fingerprint under UV 254 nm; HPTLC fingerprint under UV 366 nm; HPTLC fingerprint after derivatizing with anisaldehyde sulphuric acid reagent. Click here to view full image

Table 4. Comparative HPTLC finger print of Glycyrrhiza glabra, and Glycyrrhizin ammonical hydrate std. under UV 254, UV 366 nm and after derivatisation with anisaldehyde sulphuric acid reagent. Click here to view full image

Table 5. Comparison of HPLC peak profiles of the three G. glabra root samples. Click here to view full image

Figure 4. HPLC chromatogram of Aqueous extract of G. glabra (Bangalore) root sample. Click here to view full image

Figure 5. HPLC chromatogram of Aqueous extract of G. glabra (Bangalore) root sample. Click here to view full image

Figure 6. HPLC chromatogram of Aqueous extract of G. glabra (Nahan) root sample. Click here to view full image

Figure 7. HPLC chromatogram of standard Glycyrrhizin ammonical hydrate. Click here to view full image

Under UV 254 nm (Fig 2 UV 254), G. glabra samples showed 4 bands under UV 254 nm (Rf 0.42, 0.66, 0.79, 0.95), one band at Rf 0.42 corresponding to standard glycyrrhizin ammonical hydrate band at Rf 0.42. Under UV 366 nm (Fig 3, UV 366 nm), G. glabra samples showed 5 bands at Rf 0.19, 0.29, 0.61, 0.67, 0.82 and no band was observed for std glycyrrhizin ammonical hydrate. After derivatisation with anisaldehyde sulphuric acid reagent (Fig 4), G. glabra samples showed 5 bands at Rf 0.18 (green), 0.33 (light green), 0.42 (purple), 0.66 (yellow), 0.83 (light brown), one band at Rf 0.42 (purple) corresponding to standard glycyrrhizin ammonical hydrate band at Rf 0.42 (purple).

DISCUSSION

The present study was aimed at standardizing the different samples of Glycyrrhiza glabra collected from different markets in India. Variations in the physico-chemical values were observed but were within the prescribed limits for all the three samples (The Ayurvedic pharmacopeia of India. 1st ed., 2007). Earlier studies have reported the analysis of nine samples of Glycyrrhiza glabra from various locations of Calabria, Italy, with the aim to determine the variability in active constituents and in antibacterial and antifungal activities of the extracts.
Remarkable differences were observed in the chemical composition and biological activity. The variation of the samples, from the phytochemical and biological point of view, can be attributed to environmental factors, such as geographical coordinates, altitude and solar exposure (Giancarlo et al, 2004). In the present study, the G. glabra root samples from different locations in India revealed unique results. Saponins have been reported as one of the major components of G. glabra (Indian Herbal pharmacopoeia, 2002; Hsiang CY, 2002). In the present study the phytochemical screening and the quantification of the different extracts of the roots of G. glabra revealed the presence of saponins in the alcoholic and aqueous fractions only.
Many flavonoids such as liquiritin, liquiritigenin, isoliquiritin and isoliquiritigenin have been isolated from Spanish Glycyrrhiza species (Glycyrrhiza glabra – Monograph). We found the presence of flavonoids in the acetone, alcoholic and aqueous extracts only.

CONCLUSION

Chromatographic fingerprinting is a very unique tool for the separation and characterization of phytoconstituents (Sharma V and Janmeda P, 2013). A simple capillary-zone electrophoresis (CZE) method has been reported for G. glabra L., G uralensis Fisch. and G. inflata Bat (Leguminosae) as well as commercial licorices from Europe and China (Rauchensteiner et al, 2005).The solvent system n-butanol:acetic acid: ater (40:10:50) has been reported for the identification of Glycyrrhizic acid by TLC (Rajpal, 2002). Glycyrrhizin has been estimated earlier by TLC-densitometry using n-butanol – acetic acid – water (7:1:1) (Takino Y et al, 1979). Spots of glycyrrhizin in samples and standard were measured in the reflectance mode at 257 nm (Rajpal, 2002). We have optimized the solvent system Chloroform: methanol: glacial acetic acid: water (8: 4: 1.5:1) with respect to the standard glycyrrhizin ammonical hydrate used which gave good separation and resolution of constituents with maximum number of bands observed under UV 254, UV 366 nm and after derivatisation with anisaldehyde sulphuric acid reagent.
A high-performance liquid chromatographic (HPLC) method for measuring 18β-glycyrrhetinic acid in human plasma in the range of 0.1–3 μg/ml has been developed (Brown et al, 1991). The method employed in this study confirmed the presence of glycyrrhizin ammonical hydrate in all the samples.

CONFLICT OF INTEREST
None declared.

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