INTRODUCTION:

Ancient times, herbal remedies have been utilised to cure a wide range of illnesses. These herbal remedies are employed as their extracts and essential oils, which have been shown via multiple in vitro and in vivo investigations to have a wide range of biological qualities (Eby Aluckal, Asif Ismail,.et al., 2017). Its properties include those of an anthelminth, antibacterial, antiseptic, antifungal, antiviral, and antiprotozoal^1–6.

It was discovered to possess anti-allergic, anti-inflammatory, analgesic, anticancer, antioxidant, anticonvulsant, antidepressant, antimutagenic, and diuretic qualities^1–4,7–9. Herbal remedies have been utilised in endodontics and conservative dentistry because of these qualities^10,11.

Liquorice, sweet root or mulethi are different names for Glycyrrhiza glabra (GG)(Kaur et al., 2020). Peas and beans are its family members. Both traditional and herbal medicine and food products contain large amounts of it¹³. It is a moisturizing, wonderfully delicious herb. In Kampo medications, it is frequently utilized as a flavoring agent¹⁴. The triterpene saponin glycyrrhizin¹⁵ is said to be highly concentrated (between 2.5% and 9%) in the roots of GG (Sen, S., & Singh, R et al., 2021). Since glycyrrhetinic acid is the source of the compound's favorable characteristics, glycyrrhizin is a diglucuronide of it that also possesses antiviral, anticarcinogenic, and anti-inflammatory qualities (Akamatsu et al., n.d.2019). Patients with diabetes mellitus who take root extract from Glycyrrhiza glabra see a significant reduction in hyperglycemia and the oxidative stress caused by free iron.The Glycyrrhiza genus yields liquorice root extract^11,16, which has attracted a lot of interest due to its variety of bioactive chemicals and possible use in medicine. Studies demonstrate how it can be used to treat a range of illnesses, such as neurodevelopmental problems, metabolic abnormalities, and toxicity reduction¹⁷.

Leaf and fruit extracts from Terminalia chebula¹⁸ have strong antibacterial activity against a variety of gram-positive and gram-negative human pathogenic microorganisms (Mahesh et al., 2019). To yet, however, no research has examined the extracts' capacity to prevent the bacterial triggers of autoimmune inflammatory illnesses from growing⁸. Drug therapies that target the onset of these diseases can offer preventive chemotherapeutic alternatives. Previous research has discovered the bacterial causes of several autoimmune inflammatory diseases in genetically sensitive persons^2,3. Ankylosing spondylitis has been related to Klebsiella pneumoniae, rheumatoid arthritis has been linked to Proteus mirabilis, and multiple sclerosis has been linked to Acinetobacter baylyi and Pseudomonas aeruginosa^8,18,20. Furthermore, the potential potentiating efficacy of T. chebula extracts in combinational trials with traditional antibiotics has not yet been explored²¹.

The aim of the present study is to evaluate the Methanol, Ethanol, Water extracts (bipolar, midpolar, low polar) of two plants by comprehensively evaluate the bioactive properties of two plant extractsof phytochemical, antioxidant, antimicrobial, antifungal, anti-inflammatory and anticancer activity by MTT assay of root extract of Glycyrrhiza glabra and fruit extract of Terminalia chebula.

MATERIALS AND METHODS:

Preparation of extract:

The fruit extract of Terminalia chebula and the root extract of Glycyrrhiza glabra were gathered from the commercial market. In a conical flask, 20g of Glycyrrhiza glabra root extract was weighed and steeped in 100ml of methanol. After filtering and collecting the supernatant, light yellow was obtained for Glycyrrhiza glabra and dark yellow for Terminalia chebula. The extraction method was performed by Methanol, Ethanol and Aqueous^22,23 extract^14,21.

Phytochemical analysis:

Glycyrrhiza glabra and Terminalia chebula extracts were submitted to standard procedures for preliminary phytochemical screening and to standard reagents for phyto constituent screening²⁴. These methods of screening are employed to ascertain whether phytochemical components are present²⁵. A few plant droplets extract was dissolved in a little amount of Con. HCL, followed by a small amount of picric acid or We'll add Mayer's reagent next. Curdy or yellow precipitate was interpreted as proof that the existence of alkaloids. Few drops of extract mixed with one millilitre of methanol and one millilitre of CHCl₃ before cautiously adding con H₂SO₄ around the test tube borders^9,26. If the colour red A ring will show up in the interface, indicating that terpenoids are present²⁷. A few drops of the extracts diluted with one millilitre of methanol and a few drops of recently made FeCl₃ solution. Verify the existence of the phenol component if a green or violet hue appears¹¹. A few drops of plant extracts diluted with one millilitre of methanol and a few drops of NaOH solution will be added to the extract for the flavonoid test. If yellow colouration appears, flavonoids are present²⁸. A small amount of plant extract diluted with one millilitre of methanol and a small amount of sodium nitro prusside solution were used to produce the glycosides²⁹. Once the con.H2SO4 is added along the test tube walls, a blood red colour will develop, signifying the presence of glycosides³⁰. To perform the saponin test, dilute a few drops of plant extract with five millilitres of distilled water, and then shake the mixture violently³¹. The appearance of foam indicates the presence of saponin. A few drops of plant extract diluted with one millilitre of methanol and a few drops of acetic anhydride are added to the test tube's sidewalls, along with conc. H₂SO₄. Shake well to ensure the steroid conformation. The presence of steroids will be indicated by the appearance of various colour tints, such as green, purple, and violet³².

In-vitro antioxidant assays:

DPPH radical scavenging activity: (1,1-diphenyl-2-picrylhydrazyl)

The scavenging activity of the stable (DPPH) free radical was used to assess the antioxidant activity of methanol, ethanol, and water extract of Glycyrrhiza glabra and Terminalia chebula^2,33. One millilitre of varying doses (50–300µg/ml) of plant extract was combined with one millilitre of 0.1 mM DPPH solution in methanol, ethanol, and water. After that, the mixture was left to stand in the dark for 30 minutes. Utilising distilled water served as the benchmark. The control consisted of 1ml of DPPH solution and 1ml of methanol^30,34. The UV-Vis Spectrophotometer was used to quantify the absorbance decrease at 517nm. The following formula was used to get the inhibition percentage^9,21.

Control – Sample

% Of DPPH Radical Inhibition = -------------------- × 100

Sample

Superoxide radical scavenging activity:

Different amounts of methanol, ethanol, and water extract from Terminalia chebula and Glycyrrhiza glabra^19,35are added to the reaction mixture in that order. Additionally, 50mM of phosphate buffer (pH 7.8), 1.5 mM of riboflavin, and 12mM of NBT (Nitro Blue Tetrazolium) are also added³⁶. The reaction mixture was illuminated for fifteen minutes to initiate the reaction. The absorbance at 590nm was measured right after illumination, and the IC50 was computed. A positive control was employed, namely ascorbic acid. To get the percentage of inhibition, the following formula was used^11,21.

Control – Sample

% Of Superoxide Radical Inhibition = --------------- ×100

Sample

Phosphomolybdenum reduction assay:

The antioxidant potential of the Glycyrrhiza glabra and Terminalia chebula^11,31 methanol, ethanol, and water extracts^21,26 was evaluated according to Prieto et al. The reagent solution comprising ammonium molybdate (4 mM), sodium phosphate (28mM), and sulphuric acid (600mM) was mixed with the plant extracts at concentrations ranging from 20 to 120µg/ml. The absorbance reaction mixture was incubated for 30 minutes at 90°C in a waterbath. The coloured complex's absorbance was measured at 695nm. As a standard, distilled water was employed. The following formula was used to get the percentage of inhibition^2,9.

Sample – Control

% of Phosphomolybdenum Reduction = ----------- ×100

Sample

Ferric (fe²⁺) reducing power assay:

The method of Yen and Chen, with minor modifications, was utilized to ascertain the reducing power of Methanol, Ethanol, and Water extract of Glycyrrhiza glabra and Terminalia chebula^31,32. 1ml of phosphate buffer (0.2 M, pH 6.6) and 1ml of 1% (w/v) potassium ferricyanide [K3Fe (CN)6] were combined with 1ml of plant extracts at varying concentrations (20-120µg/ml). A millilitre of 10% (w/v) trichloroacetic acid was added to each mixture, and they were then incubated at 50°C for 30minutes^30,34. The absorbance at 700 nm was then measured using a spectrophotometer after adding 1 ml of a 0.1% (w/v) FeCl3 mixture. For standard reference, distilled water was utilized. Using the following formula, the percentage of inhibition was determined^9,21.

Sample – Control

% Of Fe ³⁺ Reduction = ------------------------------ × 100

Sample

Well diffusion Method:

Antibacterial activity:

Gram Positive strains of bacteria like Staphylococcus aureusand Bacillus subtilis, as well as Gram Negative strains like Escherichia coli, Klebsiella pneumonia,^24,37,38 were employed in the assessment of antibacterial activity³⁹. Using the agar well diffusion method, the antibacterial activity of methanol, ethanol, and water extract of Glycyrrhiza glabra and Terminalia chebula was investigated^19,35. Sterilised cotton swabs were used to disperse the inoculums onto the solidified agar medium in the petriplates. The cotton swabs were submerged on the test tube holding the inoculum before being dispersed uniformly. Using a sterile 8mm diameter well-borer, five wells were made in each plate. Subsequently, the leaf extract was added to each well with concentrations of 250, 500, and 1000µg/ml^40,41. The diameter of the inhibition zone that formed around each well on the plates containing extract-loaded wells was used to measure the antibacterial activity of the plates after they were incubated for 24hours at 37°C^2,3,39.

Antifungal activity:

The antifungal activity was evaluated through microorganisms such as Candida albicans, Candida krusei, and Candida tropicalis.Agar well diffusion method was used to test the antifungal activity of methanol, ethanol, and water extract of Glycyrrhiza glabra and Terminalia chebula^42,43. Inoculation of the solidified Potato Dextrose Agar in the petri plates was accomplished by dispensing the inoculums using sterile cotton swabs that had been previously submerged in a test tube containing the inoculums and evenly spread over the solidified agar medium⁹. Using a sterile well-borer with an 8 mm diameter, five wells were made in each plate. Next, 1 mg/ml doses of the extracts were added to each well^6,14.²¹

Anti-inflammatory activity:

Blood was drawn and placed into centrifuge tubes from a healthy volunteer who had not used any NSAIDs (non-steroidal anti-inflammatory drugs) for two weeks before the experiment^44,45. After centrifuging the tubes for 10 minutes at 3000 rpm, they were three times cleaned with an equivalent volume of regular saline^46,47. After measuring the volume of blood, 10% v/v suspension using regular saline was created.Saline was added to the control test tube in place of the test sample, and the reaction mixture (2ml) contained 1 ml of 10% RBC suspension and 1ml of test sample at various concentrations (50–300μg/ml). For thirty minutes, the reaction mixture-containing centrifuge tubes were all incubated in a water bath at 56˚C^31,32. The tubes were cooled with running tap water after the incubation period. After five minutes of centrifuging the reaction mixture at 2500rpm, the supernatant absorbance was measured at 560nm. For each test sample, the experiment was run three times. The following formula was used to get the percentage inhibition of haemolysis. The % inhibition of haemolysis was calculated using the following formula (Bhadoria et al., 2019; A. Li et al., 2021b; Na Takuathung et al., 2023).

Control – Sample

% Of inhibition = ----------------------------------- × 100

Control

Anticancer activity:

Cytotoxicity Test:

Cell viability is measured using the MTT assay, a calorimetric technique. In living cells, the tetrazole yellow MTT (3-(4,5-Dimethlthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is converted to purple formazan^39,51,52. Since the amount of viable (live) cells can be directly correlated with conversion, this decline can only occur when the enzymes responsible for mitochondrial reductase are active. There were four different extract concentrations prepared: 100ng, 10ng, 1 ng, and 1µg. In 96-well plates, the cells were cultured and subjected to varying extract concentrations. Cells are cultured for 2-4hours after being treated with MTT reagent and left for a full day. Aspiration of the medium ends the reaction, and the formazan crystals that were produced are dissolved in DMSO. The absorbance was measured at 595nm, which is directly in line with the vitality of cells⁵².

% Of cytotoxicity = (1-OD control- OD test)/ OD control*100

RESULTS:

Phytochemical Analysis of Glycyrrhiza glabra:

The Phytochemical analysis of Methanol, Ethanol and Aqueous extract of Glycyrrhiza glabra showed the presence of terpenoids, flavonoids, phenolic compounds, tannins, carbohydrates, quinones, steroids, glycosides, saponins and proteins^15,12,53

Phytochemical Analysis of Terminalia chebula:

The Phytochemical analysis of Methanol, Ethanol and Aqueous extract of Terminalia chebula^{8,13,16,27,53,54}showed the presence of Alkaloid, terpenoid, phenolic compounds, carbohydrates, quinones, steroids, glycosides and protein^{538,13,16,27,53,54.}

DPPH˙ radical scavenging assay:

One common antioxidant test is the DPPH free radical scavenging method. Using a spectrophotometer set to detect absorbance at a wavelength of 517nm, the DPPH radical scavenging assay is a decolourisations method that determines an antioxidant's ability to directly scavenge DPPH˙ radicals^24,26. The 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) was used to test the scavenging abilities of methanol, ethanol, and aqueous extracts of Glycyrrhiza glabra and Terminalia chebula^26,55. For G. glabra and T. chebula,^{8,13,16,27,53,54} the highest DPPH˞ radical scavenging activity was 67.66 ±0.95% and 80.040.95% at 120µg/mL concentration in the aqueous extract, respectively. By converting the stable DPPH (1,1-diphenyl-2-picrylhydrazyl) radical to the yellow-coloured 1,1-diphenyl-2-picrylhydrazine, aqueous extracts of Glycyrrhiza glabra and Terminalia chebula showed a strong capacity for scavenging free radicals^{8,13,16,27,53,54}. This reduction capacity improved with increasing extract concentration. Upon comparison with the standard (ascorbic acid, IC50 = 11.98μg/mL concentration), the IC50 values were determined to be 112.7μg/mL and 57.72μg/mL, respectively^2,19.

Superoxide Radical Scavenging Activity:

Superoxide anion also forms additional types of free radicals and oxidising agents, which can amplify its detrimental effects on cellular components^9,21. Because they scavenge superoxide anions, flavonoids are powerful antioxidants⁵⁶. The riboflavin-light-NBT system's production of superoxide anions from dissolved oxygen will lower NBT in this system. The yellow dye (NBT2+) in this method is reduced by superoxide anion to blue formazan, which is detected at 590nm in a UV-Vis spectrophotometer^25,35. The production of blue NBT can be inhibited by antioxidants, and a decrease in absorbance with antioxidants^11,44 suggests that superoxide anion consumption has occurred in the reaction mixture. At 120µg/mL concentration, the maximal superoxide radical scavenging activity of Glycyrrhiza glabra and Terminalia chebula was 80.95±0.40% and 76.56±0.45%, respectively, while the IC50 was 80 µg/mL and 105.6 µg/mL. The standard used for comparison was ascorbic acid (IC50 = 9.65 μg/mL concentration)^9,44.

Phosphomoybdenum Reduction Assay:

The phophomolybdenum reduction method was utilised to measure the total antioxidant activity of Methanol, Ethanol, and Aqueous extracts of Glycyrrhiza glabra and Terminalia chebula^9,44,57. This method is based on the reduction of Mo (VI) to Mo(V) by the formation of green phosphate/Mo (V) complex at acidic pH, with a maximum absorption at 695nm. At 120µg/mL concentration, the maximal phosphomolybdenum reduction^2,58was 195.51±0.75% and 134.51±0.12%, respectively, and the RC50 was 64.55µg/mL and 12.93µg/mL concentration. It was contrasted with the typical ascorbic acid concentration of 12.93μg/mL (RC50 = 45μg/mL). The PM test is a quantitative technique used to examine the rate of reduction reaction between the molybdenum ligand, oxidant, and antioxidant. It includes a longer incubation period at a higher temperature in order to thermally generate auto-oxidation^9,19,35.

Ferric (Fe²⁺) Reducing Power Assay:

The reduction of Fe3+ to Fe2+ by methanol, ethanol, and aqueous extracts of Terminalia chebula and Glycyrrhiza glabra, followed by the creation of ferro-ferric complex, was the method used for the reducing power test^2,19,58. With an increase in extract concentration, the reduction ability rises. At 120µg/mL concentration, the maximal Fe3+ reduction was 162.02±0.40% and 196.03±0.75%, respectively, and the RC50 was 49.34µg/mL and 8.5µg/mL concentration. The standard ascorbic acid concentration (RC50 = 7.72 μg/mL) was used for comparison. larger reaction mixture absorbance in this experiment also denotes a larger reduction potential. The ability of the aqueous extract to reduce serves as key evidence of its possible antioxidant activity^30,58. Utilising the Fe3+ to Fe2+ reduction assay, which shows that the extract's reducing ability varies from yellow to green or blue depending on the antioxidant content, was done. The methanol, ethanol, and aqueous extracts of Terminalia chebula^31,34and Glycyrrhiza glabra exhibited a concentration-dependent reducing capability due to the presence of antioxidants such as flavonoids and phenolic acids in significant amounts (Mutaillifu et al., 2020).

ANTIBACTERIAL ACTIVITY:

Microorganisms such as Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae) were tested for in vitro antibacterial activity using the methanol, ethanol, and aqueous extracts of Glycyrrhiza glabra and Terminalia chebula.^40,59 Through quantitative measurements of the clear zone diameter in cultures in petriplates, the extract's antibacterial sensitivity and potency were evaluated^37,38,60. A dose of 100µg/mL resulted in a maximal zone of inhibition of 21mm for Staphylococcus aureus. The presence of secondary metabolites such tannin, terpenoids, phenolic compounds, and alkaloids that inhibit microbial development could be the cause of the antibacterial activity^37,59. Microorganisms such as Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae) were tested for in vitro antibacterial activity using the methanol, ethanol, and aqueous extracts of Glycyrrhiza glabra and Terminalia chebula^40,41. Through quantitative measurements of the clear zone diameter in cultures in petriplates, the extract's antibacterial sensitivity and potency were evaluated. When Staphylococcus aureus was present at a concentration of 50 µg/mL⁵⁹, the highest zone of inhibition was 22 mm. The presence of secondary metabolites such tannin, terpenoids, phenolic compounds, and alkaloids that inhibit microbial development could be the cause of the antibacterial activity⁵⁹.

ANTIFUNGAL ACTIVITY:

The in vitro antifungal activity of Glycyrrhiza glabra, Terminalia chebula, and methanol, ethanol, and aqueous extracts against Candida albicans, Candida krusei, and Candida tropicalis was studied⁶¹. At a dose of 100 µg/mL, the greatest zone of inhibition for Candida krusei measured 19mm⁴³. In vitro antifungal activity against Candida albicans, Candida krusei, and Candida tropicalis was examined in relation to the methanol, ethanol, and aqueous extracts⁶¹ of Glycyrrhiza glabra and Terminalia chebula. By measuring the diameter of the clear zone in cultures in petriplates, the extract's antifungal sensitivity and potency were quantitatively evaluated. When the concentration of Candida krusei was 25µg/mL, the highest zone of inhibition was 20 mm.

CYTOTOXICITY ASSAY:

The MTT assay was used to assess the extracts' cytotoxicity, as previously documented. Cancer cell lines, MCF7 (human breast adenocarcinoma, ATCC-HTB22 were obtained. To ascertain the extracts' cytotoxicity and selectivity, the MCF7 cell lines underwent testing^39,62. The considerable impact of concentrations on cell viability was examined in further experiments. In 96-well plates (3 × 103/well), MCF7 cells were cultivated and incubated at 37°C for an entire night. In the initial series of tests, the samples were evaluated for cytotoxic activity at a concentration of 100 μg/mL (DMSO 0.4%; n = 3). Following a 48-hour incubation period, MTT was added to each well and the plates were left to incubate for an additional three hours^39,63.

Figure 1: Cytotoxicity Assay

CONCLUSION:

Although liquorice’s natural composition and biochemistry have been thoroughly investigated, further research is still needed to confirm the plant's ability to treat a variety of illnesses. To understand the mechanism of action, research on the several liquorice components and their biological targets is necessary. To prove the synergy between the toxicity and efficacy of other ingredients in combination treatments, more research is required. The benefits of liquorice use typically do not outweigh the possible drawbacks. For the treatment of various human ailments, liquorice extract is still widely used by many people in developing nations. Researchers have used liquorice to help create several medications. The biological significance of this herb's active ingredients has been thoroughly tested, and in vitro, in vivo, and human clinical investigations have given us strong evidence to advance our investigation. The usage of liquorice in pharmaceutical companies may rise, but it should be done so in a proper and controlled manner. A good dose plan for liquorice is necessary to heal various disorders. This analysis concluded that developing successful pharmaceutical formulations would need merging the separated phytochemical elements from liquorice with their biological significance in combating various physiological illnesses and secondary metabolites. In summary of this review, liquorice extracts and liquorice flavonoids have been used for their antibacterial, antifungal, antioxidant, and other properties.To extrapolate their mode of action in other biological functions, however, further studies are required. Along with its hepato-, cardio-, and neuroprotective qualities, myrobalan (Terminalia chebula) has considerable antioxidant and anti-inflammatory potential. It works well for disorders of the gastrointestinal system, diabetes mellitus, and cancer resurgence. To protect humans from the harmful effects of infections, the plant's antibacterial potential is also exceptionally important. In T. chebula, a variety of nutritional and phytochemical substances (such as proteins, carbohydrates, tannins, flavonoids, triterpenoid, and phenolic compounds) are formed that may have biological and pharmacological potential. All things considered, T. chebula is a great herb for enhancing personal wellness.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank Dr. M.G.R. Educational and Research Institute, Maduravoyal, Armats Biotek for their kind support during their lab studies.

REFERNCES:

1. Yu JY, Ha JY, Kim KM, Jung YS, Jung JC, Oh S. Anti-inflammatory activities of licorice extract and its active compounds, glycyrrhizic acid, liquiritin and liquiritigenin, in BV2 cells and mice liver. Molecules. 2015; 20(7): 13041-13054. doi:10.3390/molecules200713041

2. R A, P D, D G, V S. A Study on In-vitro Anti-Inflammatory and Antioxidant Activity of Aqueous Extract of Terminalia chebula. International Journal Of Pharmaceutical Quality Assurance. 2024; 15(02): 907-910. doi:10.25258/ijpqa.15.2.57

3. Bhadoria N, Gunwal MK, Suryawanshi H, Sonarkar S. Antiadherence and antimicrobial property of herbal extracts (Glycyrrhiza glabra and Terminalia chebula) on Streptococcus mutans: An in vitro experimental study. Journal of Oral and Maxillofacial Pathology. 2019; 23(1): 73-77. doi:10.4103/jomfp.JOMFP_103_18

4. Althobiti MM, Alzahrani B, Elderdery AY, et al. In vitro anti-cancer and antimicrobial effects of manganese oxide nanoparticles synthesized using the Glycyrrhiza uralensis leaf extract on breast cancer cell lines. Green Processing and Synthesis. 2023; 12(1). doi:10.1515/gps-2023-0063

5. Peng F, Du Q, Peng C, et al. A Review: The Pharmacology of Isoliquiritigenin. Phytotherapy Research. 2015; 29(7): 969-977. doi:10.1002/ptr.5348

6. Akamatsu H, Asada Y, Niwa Y. Mechanism of Anti-Inflammatory Action of Glycyrrhizin: Effect on Neutrophil Functions Including Reactive Oxygen Species Generation.

7. Hooshmandi H, Ghadiri-Anari A, Ranjbar AM, Fallahzadeh H, Hosseinzadeh M, Nadjarzadeh A. Effects of licorice extract in combination with a low-calorie diet on obesity indices, glycemic indices, and lipid profiles in overweight/obese women with polycystic ovary syndrome (PCOS): a randomized, double-blind, placebo-controlled trial. J Ovarian Res. 2024; 17(1). doi:10.1186/s13048-024-01446-9

8. Shareef THMA, Masood MD. Phytochemical and Molecular Docking Studies on Indigenous Herbs Glycyrrhiza glabra, Terminalia chebula and Hamdard joshanda. Int J Pharm Investig. 2022; 12(1): 07-14. doi:10.5530/ijpi.2022.1.2

9. Mutaillifu P, Bobakulov K, Abuduwaili A, et al. Structural characterization and antioxidant activities of a water soluble polysaccharide isolated from Glycyrrhiza glabra. Int J Biol Macromol. 2020; 144: 751-759. doi:10.1016/J.IJBIOMAC.2019.11.245

10. Batiha GES, Beshbishy AM, El-Mleeh A, Abdel-Daim MM, Devkota HP. Traditional uses, bioactive chemical constituents, and pharmacological and toxicological activities of Glycyrrhiza glabra L. (fabaceae). Biomolecules. 2020; 10(3). doi:10.3390/biom10030352

11. Martins N, Barros L, Dueñas M, Santos-Buelga C, Ferreira ICFR. Characterization of phenolic compounds and antioxidant properties of Glycyrrhiza glabra L. rhizomes and roots. RSC Adv. 2015; 5(34): 26991-26997. doi:10.1039/c5ra03963k

12. Kaur R, Kaur H, Dhindsa AS. Glycyrrhiza GLABRA: A Phytopharmacological Review. Int J Pharm Sci Res. 2013; 4(7): 2470. doi:10.13040/IJPSR.0975-8232.4(7).2470-77

13. Mamedov NA, Egamberdieva D. Phytochemical constituents and pharmacological effects of licorice: A review. In: Plant and Human Health: Pharmacology and Therapeutic Uses. Vol 3. Springer International Publishing. 2019: 1-21. doi:10.1007/978-3-030-04408-4_1

14. Hasan MK, Ara I, Mondal MSA, Kabir Y. Phytochemistry, pharmacological activity, and potential health benefits of Glycyrrhiza glabra. Heliyon. 2021; 7(6): e07240. doi:10.1016/J.HELIYON.2021.E07240

15. Graebin CS. The Pharmacological Activities of Glycyrrhizinic Acid (“Glycyrrhizin”) and Glycyrrhetinic Acid. In: Reference Series in Phytochemistry. Springer Science and Business Media B.V.; 2018: 245-261. doi:10.1007/978-3-319-27027-2_15

16. Simmler C, Pauli GF, Chen SN. Phytochemistry and biological properties of glabridin. Fitoterapia. 2013; 90: 160-184. doi:10.1016/j.fitote.2013.07.003

17. Rizzato G, Scalabrin E, Radaelli M, Capodaglio G, Piccolo O. A new exploration of licorice metabolome. Food Chem. 2017; 221: 959-968. doi:10.1016/j.foodchem.2016.11.068

18. Gupta A, Pandey S. A review on pharmacological activity of Terminalia chebula. Indian Journal of Microbiology Research. 2022; 9(3): 153-159. doi:10.18231/j.ijmr.2022.028

19. Mahesh R, Bhuvana S, Begum VMH. Effect of Terminalia chebula aqueous extract on oxidative stress and antioxidant status in the liver and kidney of young and aged rats. Cell BiochemFunct. 2009; 27(6): 358-363. doi:10.1002/cbf.1581

20. Li K, Diao Y, Zhang H, et al. Tannin extracts from immature fruits of Terminalia chebula Fructus Retz. promote cutaneous wound healing in rats. BMC Complement Altern Med. 2011;11:86. doi:10.1186/1472-6882-11-86

21. priya S, Lakshmi T, Roy A, Rajeshkumar S. Antioxidant and Antidiabetic Activity of Ethanolic Extract of Terminalia Chebula. Journal of Complementary Medicine Research. 2022;13(2):82. doi:10.5455/jcmr.2022.13.02.16

22. Pawar AR, Vikhe DN, Jadhav RS. Recent Advances in Extraction Techniques of Herbals - A Review. Asian Journal of Research in Pharmaceutical Science. 2020; 10(4): 287-292. doi:10.5958/2231-5659.2020.00050.8

23. Patel A, Kushwah P, Pillai S, Raghuvanshi A, Deshmukh N. Formulation and evaluation of herbal hand wash containing ethanolic extract of Glycyrrhiza glabra root extract. Res J Pharm Technol. 2017; 10(1): 55-57. doi:10.5958/0974-360X.2017.00013.0

24. Jayam RJ, Sivaraj C. Antioxidant, Antibacterial Activities, GC-MS Analysis of Methanol Leaves Extract in BarleriaPrionitis L. www.ijert.org

25. Varghese K, Chitra S, Abdul Azeez Sheriff S. Phytochemical Screening and In-vitro Studies on Anti-Inflammatory Properties of Sapindusemarginatus. Research J Pharm and Tech. 3(4). www.rjptonline.org

26. Vaghela JS, Sisodia SS. In Vitro Antioxidant Activity of Terminalia chebula Fruit Extracts. Research J Pharm and Tech. 2011;4(12). www.rjptonline.org

27. Meena A, Rao M, Sharma K, Yadav A, Singh U. Physicochemical and Preliminary Phytochemical Studies on the Fruit of Terminalia chebula Retz. Asian J Research Chem. 3(4). www.ajrconline.org

28. Gupta S, Bishnoi JP. Phytochemical screening of Terminalia arjuna and Glycyrrhiza glabra showing the effect of different drying techniques. Res J Pharm Technol. 2019; 12(4): 1566-1568. doi:10.5958/0974-360X.2019.00259.2

29. Gupta S, Bishnoi JP. Phytochemical screening of Terminalia arjuna and Glycyrrhiza glabra showing the effect of different drying techniques. Res J Pharm Technol. 2019; 12(4): 1566-1568. doi:10.5958/0974-360X.2019.00259.2

30. Na Takuathung M, Wongnoppavich A, Jaijoy K, Soonthornchareonnon N, Sireeratawong S. Antioxidant and Antitumorigenic Activities of the Standardized Water Extract From Fruit of Terminalia chebula Retz. var. chebula. Nat Prod Commun. 2023; 18(6). doi:10.1177/1934578X231176925

31. Tohma HS, Gulçin I. Antioxidant and radical scavenging activity of aerial parts and roots of Turkish liquorice (Glycyrrhiza glabra L.). Int J Food Prop. 2010; 13(4): 657-671. doi:10.1080/10942911003773916

32. Naidu N, Kumar GS, Sivakrishna K, Anjinaik K, Kumar LP, Sneha G. Anti microbial and antioxidant evolution of aqueous extract of Terminalia chebula using disc diffusion, H2O2 scavenging methods .Asian Journal of Research in Pharmaceutical Science. 2017;7(2):112. doi:10.5958/2231-5659.2017.00017.0

33. Konduri MKR, Bogolu VR. Evaluation of antioxidant activities of two medicinal plants, terminalia chebula and adhatodavasica belonging to Bapatla, India. Res J Pharm Technol. 2015; 8(2): 194-197. doi:10.5958/0974-360X.2015.00035.9

34. Konduri MKR, Bogolu VR. Evaluation of antioxidant activities of two medicinal plants, terminalia chebula and adhatodavasica belonging to Bapatla, India. Res J Pharm Technol. 2015; 8(2): 194-197. doi:10.5958/0974-360X.2015.00035.9

35. Paliwal R, Sharma V, Sharma S. Elucidation of Free Radical Scavenging and Antioxidant Activity of Aqueous and Hydro-Ethanolic Extracts of Moringa oleifera Pods. Research J Pharm and Tech. 2011; 4(4). www.rjptonline.org

36. Vaghela JS, Sisodia SS. In Vitro Antioxidant Activity of Terminalia chebula Fruit Extracts. Research J Pharm and Tech. 2011; 4(12). www.rjptonline.org

37. Haraguchi H, Tanimoto K, Tamura Y, MizutanitK!, Kinoshita~ T. ~ Pergamon Mode Of Antibacterial Action Of Retrochalcones From Gl Ycyrrhiza Inflata. Vol 48.; 1998.

38. Amer SA, Abushady HM, Refay RM, Mailam MA. Enhancement of the antibacterial potential of plantaricin by incorporation into silver nanoparticles. Journal of Genetic Engineering and Biotechnology. 2021;19(1). doi:10.1186/s43141-020-00093-z

39. Althobiti MM, Alzahrani B, Elderdery AY, et al. In vitro anti-cancer and antimicrobial effects of manganese oxide nanoparticles synthesized using the Glycyrrhiza uralensis leaf extract on breast cancer cell lines. Green Processing and Synthesis. 2023; 12(1). doi:10.1515/gps-2023-0063

40. Shah CP, Joshi VJ, Patel DM, et al. Evaluation of In Vitro Antibacterial Activity of Roots of Glycyrrhiza glabra Against Multidrug Resistant Bacterial Pathogens of E. coli. Research J Pharm and Tech. 2011; 4(4). www.rjptonline.org

41. Shah CP, Joshi VJ, Patel DM, et al. Evaluation of In Vitro Antibacterial Activity of Roots of Glycyrrhiza glabra Against Multidrug Resistant Bacterial Pathogens of E. coli. Research J Pharm and Tech. 2011; 4(4). www.rjptonline.org

42. Kusirisin W, Srichairatanakool S, Lerttrakarnnon P, et al. Antioxidative Activity, Polyphenolic Content and Anti-Glycation Effect of Some Thai Medicinal Plants Traditionally Used in Diabetic Patients. Med Chem (Los Angeles). 2009; 5(2): 139-147. doi:10.2174/157340609787582918

43. Venkatachalam P, Chittibabu CV. Antifungal activity of Terminalia chebula fruit extracts. Current Botany. Published online December 20, 2020: 216-220. doi:10.25081/cb.2020.v11.6499

44. Vaya J, Belinky PA, Aviram M. Antioxidant Constituents From Licorice Roots: Isolation, Structure Elucidation And Antioxidative Capacity Toward Ldl Oxidation. Vol 23.; 1997.

45. Biondi DM, Rocco C, Ruberto G. New dihydrostilbene derivatives from the leaves of Glycyrrhiza glabra and evaluation of their antioxidant activity. J Nat Prod. 2003; 66(4): 477-480. doi:10.1021/np020365s

46. Venkatachalam P, Chittibabu CV. Antifungal activity of Terminalia chebula fruit extracts. Current Botany. Published online December 20, 2020: 216-220. doi:10.25081/cb.2020.v11.6499

47. Li A, Zhao Z, Zhang S, Zhang Z, Shi Y. Fungicidal activity and mechanism of action of glabridin from glycyrrhiza glabra l. Int J Mol Sci. 2021; 22(20). doi:10.3390/ijms222010966

48. Dwivedi S, Dwivedi A, Kapadia R, Kaul S. Anthelmintic Activity of Alcoholic and Aqueous Extract of Fruits of Terminalia Chebula Retz. Vol 12.; 2008.

49. Li A, Zhao Z, Zhang S, Zhang Z, Shi Y. Fungicidal activity and mechanism of action of glabridin from glycyrrhiza glabra l. Int J Mol Sci. 2021; 22(20). doi:10.3390/ijms222010966

50. Na Takuathung M, Wongnoppavich A, Jaijoy K, Soonthornchareonnon N, Sireeratawong S. Antioxidant and Antitumorigenic Activities of the Standardized Water Extract From Fruit of Terminalia chebula Retz. var. chebula. Nat Prod Commun. 2023; 18(6). doi:10.1177/1934578X231176925

51. Jiang D, Rasul A, Batool R, et al. Potential Anticancer Properties and Mechanisms of Action of Formononetin. Biomed Res Int. 2019; 2019. doi:10.1155/2019/5854315

52. Ahmad R, Alqathama A, Aldholmi M, et al. Biological Screening of Glycyrrhiza glabra L. from Different Origins for Antidiabetic and Anticancer Activity. Pharmaceuticals. 2023; 16(1). doi:10.3390/ph16010007

53. Hasan MK, Ara I, Mondal MSA, Kabir Y. Phytochemistry, pharmacological activity, and potential health benefits of Glycyrrhiza glabra. Heliyon. 2021; 7(6): e07240. doi:10.1016/J.HELIYON.2021.E07240

54. Gupta S, Bishnoi JP. Phytochemical screening of Terminalia arjuna and Glycyrrhiza glabra showing the effect of different drying techniques. Res J Pharm Technol. 2019; 12(4): 1566-1568. doi:10.5958/0974-360X.2019.00259.2

55. Elias J, Varkey IC. Terminalia chebula Retz. Stem Bark Extract: A Potent Natural Antioxidant. 2011; 4(3). www.ajrconline.org

56. Jayam JR, Rajendran AK, Suganthi M, Palanisamy S. Insights Into The Roles Of Antioxidants In Citrus Fruits. Int J Pharm Sci Res. 2023; 14(3): 1098-1107. doi:10.13040/IJPSR.0975-8232.14(3).1098-07

57. Elias J, Varkey IC. Terminalia chebula Retz. Stem Bark Extract: A Potent Natural Antioxidant. 2011; 4(3). www.ajrconline.org

58. Olchowik-Grabarek E, Czerkas K, Matchanov AD, et al. Antibacterial and Antihemolytic Activity of New Biomaterial Based on Glycyrrhizic Acid and Quercetin (GAQ) against Staphylococcus aureus. J FunctBiomater. 2023; 14(7). doi:10.3390/jfb14070368

59. Jayam RJ, Sivaraj C. Antioxidant, Antibacterial Activities, GC-MS Analysis of Methanol Leaves Extract in Barleria Prionitis L. www.ijert.org

60. Li A, Zhao Z, Zhang S, Zhang Z, Shi Y. Fungicidal activity and mechanism of action of glabridin from glycyrrhiza glabra l. Int J Mol Sci. 2021; 22(20). doi:10.3390/ijms222010966

Received on 03.10.2024 Revised on 12.02.2025

Accepted on 15.04.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):38-44.

DOI: 10.52711/0974-360X.2026.00006

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.