Nutritional and therapeutic potential of Glycyrrhiza glabra L. roots

This study was aimed to estimate the antioxidant, anti-diabetic, anti-amnesic, anti-biofilm, thrombolytic, hemolytic, and cytotoxic effects of G. glabra root extracts for nutritive and medicinal purpose. Antioxidant, alpha amylase, antiglycation, anti-amnesic, cytotoxic, thrombolytic and antibiofilm assays were performed. In the current research, G. glabra root extracts showed significant antioxidant (80.96 mg GAE/100 g TPC, 38.96 mg CE/100 g TFC, 46% DPPH scavenging), antidiabetic (75% antiglycation, 38.7% α-amylase inhibition), anti-amnesic (5.5%), hemolytic (19.46%), thrombolytic (35.4%) and antibiofilm (72% P. multocida, 51.57% S. aureus inhibition) activities. G. glabra root extracts have remarkable activity to fight against oxidative stress, hyperglycemia, blood coagulation, bacterial growth, DNA damage and memory enhancing ability that ensure its uses in food and pharmaceutical industries.


Cytotoxic potential 2.5.1. DNA damage protection assay
For DNA damage protection assay, reaction mixture (supercoiled pBR322 plasmid DNA and extracts) was incubated, following the addition of Fenton reagent and analyzed by agarose gel electrophoresis. The positive control contained Fenton reagent and plasmid DNA whereas plasmid DNA was used as negative control. The results were analyzed by trans-illuminator (Ruma et al., 2013).

Hemolytic activity
This assay was performed according to described procedure (Powell et al., 2000). Red blood cells (RBCs) suspension were mixed with fractions, incubated for 30 minutes at 37°C, centrifuged at 1500 rpm for 5 minutes and supernatant was diluted with phosphate buffer saline. The absorbance was determined at 576 nm.

Thrombolytic activity
Thrombolytic activity was carried out by taking micro-centrifuge tubes. These were weighed empty, added with blood samples and incubated. Serum was discarded and tubes with clot were again weights. Clots were added with plant extracts and incubated. After incubation, tubes were again weighed see the difference in weight after clot disruption (Hossain et al., 2012).

Biofilm inhibition assay
Antibiofilm activity was investigated by using two bacterial strains namely Staphylococcus aureus and Pasteurella multocida. Plant extracts, nutrient broth and microbial strains were incubated for 24 hr. at 37ºC in microtiter plates. Then plates were washed with sodium phosphate buffer saline pH 7.4 and staining was done by crystal violet dye. Absorbance was taken at 630 nm (Dheepa et al., 2011).

Statistical analysis
The results were expressed as mean ± standard deviation (S.D) and analyzed by SPSS software (version 22) with level of significance set at p<0.05.

Results and Discussion
3.1. Anti-oxidant activity TPC, TFC and DPPH assay investigations of all fractions showed significant (p<0.05) results. Chloroform fraction showed highest phenolic contents (80.96 ± 0.019 mg GAE). TPC results for other fractions in descending order were as <methanol<ethanol<ethyl acetate<n-butanol <aqueous<n-hexane. In current research, TPC contents were in range of 3.14-80.96 mg GAE which are in accordance to earlier reported results. An earlier study reported the TPC in range of 75.7 to 185.7 μg GAE for aqueous and ethanolic extracts of G. glabra aerial parts, respectively (Tohma and Gulcin, 2010). Our results are comparatively similar to these earlier findings, although variations may be due to different solvents used and units of measurement used. Maximum flavonoid contents were shown by chloroform extract (38.96 ± 0.017 mg CE). TFC values for other fractions were found to be in ascending order as: aqueous >n-butanol>ethyl acetate>methanol> ethanol>nhexane. Flavonoids possess the antioxidant activities that are important for various diseases produced by free radicals. In present study G. glabra fractions exhibited TFC in range of 5.13-38.96 mg CE. Contrary to present results an earlier study described the 2.6-5.1μg quercitin flavonoid contents (Tohma and Gulcin, 2010). Variation among results is due to use of diverse TFC units. Optimum DPPH inhibition (46%) was shown by n-butanol. Whereas other fractions showed the following DPPH inhibition indescending order: n-hexane <aqueous<ethyl acetate<ethanol <chloroform<methanol (Table  1). G. glabra root extracts showed DPPH scavenging activity in range of 5.08-46%. Current results are in accordance with the previous investigation that reported antioxidant activity of G. glabra roots in range of 42.9-57.5 % (Tohma and Gulcin, 2010). It is concluded from the antioxidant profile that G. glabra roots have potential to eliminate free radicals to be used in food industries for preservation of food and to avoid the food spoilage and rancidity. It also protects the biological systems to minimize the detrimental effects of oxidative stress.

Antidiabetic potential
Range of antiglycation values was 35-75%; p <0.05 and trend in descending order was: chloroform <ethyl acetate<n-hexane <aqueous <methanol<ethanol<n-butanol (Table 1). Restriction of protein glycosylation process is a vital therapeutic option to reduce diabetic complications. G. glabra extracts exhibit up to 75% antiglycation potential. Another study reported the 69% glycation inhibition from G.glabra roots owing to the presence of triterpenoid saponin and glycyrrhizin that reduced level of advanced glycation end products (Tupe et al., 2017). Significant (p<0.05) alpha amylase inhibition ranged from 7.2 to 38.7%. Inhibition percentage for all fractions in descending order was as: n-hexane <chloroform<ethyl acetate <n-butanol <ethanol <methanol <aqueous (Table 1). About 7.2-38.7% inhibition potential shown by G. glabra root extracts in current research was supported by earlier study that stated amylase inhibition range from 24.12 to 51.07% (Rodda et al., 2014).Findings of these results suggested that G. glabra root extracts are valuable alternatives sweetening agent used in food products to avoid diabetic complications. Data expressed as (mean ± SD) or percentage inhibition. All the extracts showed statistically significant results p<0.05. TPC: total phenolic contents expressed mg gallic acid equivalents /100 g dry weight; TFC: total flavonoid contents expressed as mg catechin equivalents/100 g dry weight; DPPH: 2, 2-diphenyl l-picrylhydrazyl. Control: BHT; butylated hydroxy toluene (antioxidant), metformin (antiglycation), glucobay (alpha amylase inhibition).

Anti-amnesic activity
Maximum acetylcholinesterase (AChE) inhibitory potential (5.5%) was shown by ethanol fraction. Whereas other extracts showed inhibition in ascending order as: ethyl acetate >aqueous> methanol >n-butanol >chloroform >n-hexane (Table 2). AChE inhibition range shown by G. glabra was 3.12-5.5%. An earlier investigation (Young et al., 2013) perceived that glabridin containing aqueous extract suppressed the neuron cell death by inhibiting the AChE activity up to 11.1%. As AChE enzyme is involved in the termination of impulse transmission by rapid hydrolysis of the neurotransmitter acetylcholine. Therefore, current study demonstrated the impressive ability of G. glabra root extracts to regulate hormonal imbalancement involved in neurodegenerative disorders.

Cytotoxic potential 3.4.1. DNA damage protection assay
Chemical constituents of G. glabra organic extracts have the potential to protect the DNA. As shown in Figure  1, n-butanol, n-hexane and aqueous fractions have no ability to protect DNA from Fenton-induced damaged besides other fractions (methanol, ethanol, ethyl acetate, chloroform) which protected DNA from distortion ( Figure 1). Cell life cycle is affected by damage to nucleic acids, which have been linked to cell cycle regulation and repair pathways. Pretreatment of plasmid DNA with G. glabra root extracts and fractions secured DNA with similar efficacies, as four fractions (methanol, ethanol, ethyl acetate, chloroform) saved DNA from oxidative stress. Similar effect was attributed by glycyrrhetinic acid component of Glycyrrhiza uralensis that exhibit 20-80% protection to DNA (Chen et al., 1994). It is concluded that G. glabra root extracts provide encouraging results against DNA alterations associated with positive health effects. Figure 1 (a-b). Agarose gelelectrophoresis of DNA-damage protection assay. a: Lane 1: ladder; Lane 2: positive control; Lane 3: negative control (FeSO 4 ); Lane 4 and 5: negative control (H 2 O 2 ), b: Lane 1-7: plasmid DNA incubated with methanol, ethanol, n-butanol, n-hexane, chloroform, ethyl acetate and aqueous extracts respectively.

Hemolytic activity
Cytotoxic potential of G. glabra was investigated against human red cells through hemolytic assay. Ethyl acetate (19.46%) being the most active fraction in hemolysis. Antihemolytic potential of other fractions in ascending order was as: ethanol> methanol> aqueous> chloroform >n-butanol >n-hexane (Table 2). Hemolysis is mainly caused by oxidative stress that leads to erythrocytes lysis. G. glabra root extracts had up to 19.46% hemolytic ability. Current results are in agreement with the previous research that elucidated the 16% antihemolytic potential of G. glabra root extracts against human erythrocytes (Kim et al., 2002). The cytoprotective efficacy of G. glabra root ingredients has future prospects in minimizing the hemolytic impediments.

Thrombolytic activity
Highest clot distortion activity was (35.4%) exhibited by methanol extract and rest of the fractions showed descending order as: chloroform <aqueous<n-butanol <ethyl acetate< ethanol<n-hexane (Table 2). Cardiovascular disorders are mostly caused by blood clot or thrombus formation at an alarming rate. Uncontrolled platelet accumulation can be life threatening. Anticoagulation potential of G. glabra root extracts was up to 35.5%. Literature survey indicated impressive thrombus inhibition (34.8%) by glycyrrhizin (Francischetti et al., 1997). The present optimistic results reflect the usage of G. glabra root in form of herbal tincture as cardio protective and sport nutrition formulas.
Biofilm are microbial colonies adhering to a specific place that cause persistent chronic and tenacious infections. Discovery of natural antimicrobial agents is needed to lessen their detrimental complexities. Optimum growth inhibition (72%, 51.57%) was depicted by G. glabra root extracts against P. multocida and S. aureus. Available data indicates no similar approach of evaluating anti-bacterial activity of G. glabra root extracts using P. multocida and S. aureus strains. Current dynamic results unveiled promising future of G.glabra root extracts to be used in toothpaste, cosmetics, chewing gum, beverages and in food items by improving the food safety assurance for consumers by killing infectious agents. Data is presented as percentage.

Conclusions
On the basis of current study it is recommended that G. glabra root bioactive constituents are applicable to improve the food quality, safety, texture, taste, aroma and flavor. Since these G. glabra root extracts are well tolerated, relatively inexpensive and readily available, could provide complementary therapy in treatment of various diseases. However there is a need to further explore its bioactive constituent's to enhance human health status using animal trial.