Description:FIELD OF THE INVENTION
The present invention relates to a method of isolating an active compound and a pharmaceutical composition thereof for Arthritis. More particularly, the present invention relates to extraction, isolation and purification of active ingredients from the extracts prepared from Glycyrrhiza glabra, also called as Licorice. The present invention also relates to a pharmaceutical composition comprising of extracts of Glycyrrhiza glabra or isolated purified Glabridin and a method of formulation of said pharmaceutical composition for therapeutic use in Arthritis.

BACKGROUND OF THE INVENTION
Liquorice or Licorice is the common name of Glycyrrhiza glabra, a flowering plant of the bean family Fabaceae, from the root of which a sweet, aromatic flavoring can be extracted. The liquorice plant is an herbaceous perennial legume native to Western Asia, North Africa, and Southern Europe. It is found growing wild in many Asian countries and is also cultivated at small scales in India, Iran, Afghanistan etc. The root tastes sweet and is used in Ayurveda.

This fortifying and replenishing herb was utilized by ancient Egyptians and Greeks, is considered an herbal staple in Traditional Chinese Medicine (TCM) and is also a revered herb in the Ayurvedic apothecary. Derived from the roots of a flowering perennial plant that is a part of the legume family, Licorice is known scientifically as Glycyrrhiza glabra and also carries the nickname, yasthimadhu, in Ayurvedic medicine, which literally translates to "sweet stick".

Ayurveda considers this herb to be a rasayana which act as supportive tonics to reduce tiredness and fatigue; therefore, restoring strength and vitality.

Glabridin as therapeutic agent for various ailments
Glabridin is a one of the most studied biological markers of Liquorice (Glycyrrhiza glabra/ G.glabra) associated with wide range of biological properties such as antioxidant, anti-atherogenic, estrogenic, skin-whitening, anti-inflammatory, neuroprotective, anti-osteoporotic and regulation of energy metabolism. Chemically, Glabridin is a prenylated isoflavonoid mainly found in root extract of Glycyrrhiza glabra. Glabridin has shown inhibitory activities against tyrosinase enzyme and Human cytochrome P450S 3A4, 2B6 and 2C9. Glabridin induces glucose uptake through the adenosine monophosphate-activated protein kinase and may possess therapeutic effect in metabolic disorders such as diabetes and hyperglycemia by modulating glucose metabolism. Glabridin has demonstrated hyperglycemic effect in animal models of diabetes mellitus by significantly increasing body weight, glucose tolerance, and dismutase activities and decreasing fasting blood glucose in liver, kidney and pancreas.

Arthritis
Arthritis is the name of a medical condition indicated by the swelling and tenderness of one or more joints. The main symptoms of arthritis are joint pain and stiffness, which normally aggravate with age. The most common categories of Arthritis are Osteoarthritis (OA) and Rheumatoid Arthritis (RA). In Osteoarthritis, the hard slippery tissue called cartilage that covers the ends of bones where they form a joint, breaks down. Rheumatoid Arthritis is a disorder in which the immune system attacks the joints, beginning with the lining of joints. Infections or underlying diseases like Psoriasis or Lupus can trigger other types of Arthritis. Remedies for Arthritis vary depending on its type. The main objectives of arthritis therapies are to reduce symptoms of pain and stiffness to improve quality of life.

Glucose imbalance leads to arthritis.
OA is a degenerative joint disease, which is characterized by articular cartilage degeneration and loss accompanied by subchondral osteosclerosis and osteophyte formation. A recent study reported that degeneration of cartilage may be triggered by metabolic disorders of glucose balance. RA is a chronic systemic autoimmune disease featured with painful joint inflammation and disability. The changes in glucose metabolism are believed to contribute to disease progression of RA.

Hexokinase (HK) as target
Recently published studies have shown that targeting the first step in glucose metabolism, HK2, could be a possible therapeutic target for Rheumatoid arthritis fibroblast-like synoviocytes (RAFLS).

Hexokinase is a group of intracellular enzymes that phosphorylates hexose sugars such as glucose, fructose and mannose to their corresponding hexose-6-phosphates. Glucose has central metabolic importance in all organisms from microbes to humans and glycolysis is the major catabolic pathway that generates energy (ATP) by catabolism of glucose. Hexokinases plays an important role in glucose metabolism as it is the first enzyme involved in glycolytic pathway; catalyzes the phosphorylation of glucose to glucose-6-phosphate which is one of the rate limiting of glycolysis. Hexokinases (HK) have been found in every organism ranging from bacteria, yeast, and plants to humans and other vertebrates. They are grouped as actin fold proteins, sharing a common ATP binding site core and surrounded by more variable sequences that determine substrate affinities. Several isoforms of hexokinases can be present in single species providing different roles based on the location where it is expressed. In mammalian tissues, there are four major hexokinases which are HK1, HK2, HK3, and HK4. HK1 and HK2 are the main hexokinases that have a contribution in cell survival.

Glucokinase or hexokinase IV is a predominant enzyme expressed in liver and plays an important role in homeostasis of blood glucose level due to effective control on hepatic glucose disposal.

Various non-patent literatures have been published in this area of sciences however, these have not been able to effectively solve the problems associated with rheumatoid arthritis with an herbal composition with no side effects.

The publication titled “Expression of hexokinase II and Glut-1 in untreated human breast cancer”, published in 2002 and authored by Brown et al, found that breast cancers were HK2 positive in 79% of studied tumors. HK2 status in breast cancer tissue sections was significantly related to poor prognosis and relapse of breast cancers. Also, tamoxifen-resistant breast cancer cells MCF-7 showed upregulation of HK2 and mTOR that was accompanied by an enhanced glycolysis process. Furthermore, HK2 overexpression in ovarian cancer cells induces cisplatin resistance. Therefore, it was found that targeting HK2 will block glucose metabolism in cancer cells, which may inhibit its proliferation with minimum side effects reported. In prior arts so far HK inhibition has been studied as a target in cancer therapy and other viral infections only, but it has not been disclosed as an additional important marker which can be useful for treating/mitigating problems related to arthritis.

Another publication titled “New natural inhibitors of hexokinase 2 (HK2): Steroids from Ganoderma sinense”, published in 2018, has highlighted HK2 as a target in cancer therapy. The drugs such as 2-deoxyglucose, 3-bromopyruvate and metformin are found to be HK2 inhibitors. However, these chemical formulations are not effective and have severe side effects. Therefore, prior art in the existing state of art discloses the inefficient use of these chemicals in anticancer therapy. Accordingly, in spite of major advances in the treatment of RA and cancers, there is still need for convenient, safe, and effective therapies for RA and cancers.

One more publication titled “Effect of Glycyrrhiza glabra on Antigen Induced Arthritis in Mice Model” published in 2015 authored by Abdul kareem et al, studied the effect of aqueous extract of G.glabra on antigen induced arthritis model in mice. It was found that G. glabra was effective in inhibiting histopathological changes in arthritis (inflammatory cells infiltration, synovitis, cartilage erosion, bone erosion, loss of joint architecture and pannus formation) in dose dependent manner. However, the aqueous extract contains less than 1% Glabridin and contains Glycyrrhizinic acid majorly (almost 10%). As the concentration of Glabridin is very less its contribution towards inhibiting histopathological changes and causing anti arthritic effect is negligible. Therefore, the effect of Glabridin towards improving the conditions of Arthritis is not explored in this prior art.

Another publication titled as “Anti-arthritic and cartilage damage prevention via regulation of Nrf2/HO-1 signaling by Glabridin on osteoarthritis” of 2021 authored by Xiaoxu Wang et al, described the anti-arthritic mechanism of Glabridin in IL-1b induced chondrocytes and monosodium-iodoacetate (MIA) induced osteoarthritis in rat models. However, it does not provide for the effect of Glabridin on Rheumatoid arthritis or its contribution in the Hexokinase inhibition activity.

HK for arthritis diagnosis/ marker
The present invention verified that increased HK2 levels are a prospective candidate marker for RA diagnosis and correlate positively with disease activity in RA patients. Dysregulation of HK2 may participate in the molecular mechanism of RA and be an attractive selective metabolic target for RA treatment and suggest that HK-I and HK-II are directly involved in the pathogenesis of RA. Furthermore, silencing HK-I and HK-II, especially HK-II, disrupts the proliferative and inflammatory phases of RA.

Efficacy of Glabridin in hexokinase inhibition
In the present invention, the inhibitory efficacy of Glabridin on hexokinase was studied using an in-vitro hexokinase assay. The molecular mechanism of interaction between Glabridin and hexokinase enzyme was predicted using molecular docking techniques. The present invention relates to the effectiveness of Glabridin as hexokinase inhibitor. Both in-vitro assays and molecular docking study have shown that Glabridin can be used as a potent natural inhibitor of hexokinase with better stability and higher bioactivity and hence, can be used as a potential therapeutic agent for Arthritis.

Accordingly, the present invention provides a unique method of isolating active herbal compounds from Licorice and formulating a pharmaceutical composition from said active herbal compound for improving Arthritis. Said method of isolation provides a higher yield of the component obtained from Licorice.

Said pharmaceutical composition is prepared by mixing said herbal component with various pharmaceutical excipients/additives. The said pharmaceutical composition is effective and efficacious in improving the Arthritis conditions.

Therefore, the present invention overcomes the drawbacks of state of art by providing an herbal composition for improving the symptoms/conditions associated with Arthritis. Since the present composition is herbal in nature therefore, it has minimum or no side effects. Also, the present invention provides a cost-effective treatment for Arthritis.

OBJECT OF THE INVENTION
The main object of the present invention is to provide a method of extracting, isolating and purifying an active compound and a pharmaceutical composition thereof for Arthritis.

Another object of the present invention is to provide a method of extracting, isolating and purifying Glabridin from G glabra and a pharmaceutical composition thereof for improving the conditions of Arthritis.

Yet another object of the present invention is to provide a method of extracting, isolating and purifying Glabridin which is economical and provides a better yield.

Yet another object of the present invention is to provide a pharmaceutical composition which is efficient and effective for improving conditions of Arthritis.

Yet another object of the present invention is to provide a pharmaceutical composition comprising extracts of Glycyrrhiza glabra or isolated purified Glabridin, which is efficient and effective for improving conditions in Arthritis.

Yet another object of the present invention is to provide cost effective method for mitigating conditions of Arthritis.

Yet another object of the present invention is to provide an herbal pharmaceutical composition for Arthritis which has minimum or no side effects.

SUMMARY OF THE INVENTION:
Accordingly, the present invention relates to a method of extracting, isolating and purifying an active compound and a pharmaceutical composition thereof for improving conditions of Arthritis.

A versatile Ayurvedic herb, Licorice is used in gastritis, aphrodisiac therapy, fever and bleeding disorders. There are more than 300 different compounds in Licorice, some of which have anti-inflammatory, antiviral, and antimicrobial properties. For example, one animal study showed that glycyrrhizin extract from Licorice root may relieve symptoms associated with eczema. Glycyrrihiza roots contain glycyrrhizin, asparagin, sugar, starch, acid resin, gum, mucilage, phosphoric, sulfuric & malic acids. Bark contains a small quantity of tannins. The two main components of Glycyrrhiza glabra are Glabridin and glycyrrhizin (glycyrrhizic acid).

Accordingly, the present invention relates to extracting, isolating and purifying of active compound, Glabridin, from Licorice extract, which has potential for improving conditions associated with Arthritis. The present invention provides a novel method for extracting, isolating and purifying of active compound, Glabridin with high yield from Licorice.

Molecular docking studies have been carried out of Glabridin with a few arthritic marker enzymes/proteins namely, Mitogen-activated protein kinase 1, Cellular tumor antigen P53 and RAC-alpha serine/threonine-protein kinase. Pure Glabridin has been observed to have hexokinase inhibition activity, as seen in experimental work done by the inventors. Hexokinase inhibitors are known to be useful for arthritis. Hence the potency of Glabridin was examined in arthritis induced animals wherein Glabridin was observed to have potent anti-arthritic activity. The method to purify Glabridin from Licorice as well as the method to manufacture Licorice to achieve Licorice extract with optimal Glabridin were optimized. The HPLC method for Glabridin estimation was also standardized.

The present invention discloses the usefulness of Licorice for arthritis treatment. The invention also discloses about Glabridin inhibiting human hexokinase activity which is promising and is a major contribution in the field of Arthritis treatment.

The present invention relates to an extraction procedure which resulted in several fold higher yield of Glabridin. Said yield is more than 6% to 7 % than the commercially available Licorice extract. Accordingly, the present invention provides a novel method of isolating higher yields of herbal compound Glabridin from Glycyrrhiza glabra.

The present invention also relates to a pharmaceutical composition comprising Glabridin and along with suitable excipients/adjuvants. Said pharmaceutical composition is efficacious and effective for improving the conditions of Arthritis.

Said pharmaceutical composition can be administered via oral or injectable route.

Also, the present invention provides an herbal pharmaceutical composition with minimum or no side effects.

The present application provided ample of experimental data demonstrating the efficacy of pharmaceutical composition comprising Glabridin and other excipients in improving the conditions of Arthritis in animals.

BRIEF DESCRIPTION OF DRAWINGS
Figure 1 displays the Standard solution Chromatogram.
Figure 2 displays the Isolated Glabridin Chromatogram.
Figure 3 displays the Comparison of HK activity of 2-deoxy-2-glucose, licorice DPI and pure Glabridin and Glycyrrhizin.
Figure 4 displays the Molecular docking of Mitogen-activated protein kinase 1 binding with Favipiravir showing 3D model of the interactions and the 2D interaction patterns and H-bond interaction.
Figure 5 displays the Molecular docking of Mitogen-activated protein kinase 1 binding with Glabridin showing 3D model of the interactions and the 2D interaction patterns and H-bond interaction.
Figure 6 displays the Molecular docking of Mitogen-activated protein kinase 1 binding with Ivermectin showing 3D model of the interactions and the 2D interaction patterns and H-bond interaction.
Figure 7 displays the Molecular docking of Cellular Tumor Antigen P53 binding with Favipiravir showing 3D model of the interactions and the 2D interaction patterns and H-bond interaction.
Figure 8 displays the Molecular docking of Cellular Tumor Antigen P53 binding with Glabridin showing 3D model of the interactions and the 2D interaction patterns and H-bond interaction.
Figure 9 displays the Molecular docking of Cellular Tumor Antigen P53 binding with Ivermectin showing 3D model of the interactions and the 2D interaction patterns and H-bond interaction.
Figure 10 displays the Molecular docking of RAC-Alpha Serine/Threonine-protein kinase binding with Favipiravir showing 3D model of the interactions and the 2D interaction patterns and H-bond interaction.
Figure 11 displays the Molecular docking of RAC-Alpha Serine/Threonine-protein kinase binding with Glabridin showing 3D model of the interactions and the 2D interaction patterns and H-bond interaction.
Figure 12 displays the Molecular docking of RAC-Alpha Serine/Threonine-protein kinase binding with Ivermectin showing 3D model of the interactions and the 2D interaction patterns and H-bond interaction.
Figure 13 displays the Effect of SAVA 18 on Body weight (g) in FCA induced arthritis in rats.
Figure 14 displays the Effect of SAVA 18 on Paw Volume (ml) in FCA induced arthritis in rats.
Figure 15 displays the Effect of SAVA 18 on Pain Threshold (g) in FCA induced arthritis in rats.
Figure 16 displays the Effect of SAVA 18 on CRP (mg/L) in FCA induced arthritis in rats.
Figure 17 displays the Effect of SAVA 18 on ESR (mm/h) in FCA induced arthritis in rats.
Figure 18 displays the Effect of SAVA 18 on SGPT (U/L) in FCA induced arthritis in rats.
Figure 19 displays the Effect of SAVA 18 on SGOT (U/L) in FCA induced arthritis in rats.
Figure 20 displays the Effect of SAVA 18 on ALP (U/L) in FCA induced arthritis in rats.
Figure 21 displays the Effect of SAVA 18 on Liver SOD level in FCA induced arthritis in rats.
Figure 22 displays the Effect of SAVA 18 on Liver GSH level in FCA induced arthritis in rats.

DETAILED DESCRIPTION OF THE INVENTION ILLUSTRATIONS AND EXAMPLES
While the invention has been disclosed with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made, and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt to a particular situation or material to the teachings of the invention without departing from its scope.

Throughout the specification and claims, the following terms take the meanings explicitly associated herein unless the context clearly dictates otherwise. The meaning of “a,” “an”, and “the” include plural references. Additionally, a reference to the singular includes a reference to the plural unless otherwise stated or inconsistent with the disclosure herein.

The biological materials used in the present invention have been sourced as per the details given in Table 1 below.

Table 1: List of sources of biological material.
Ser No. Biological Material Location
1 Glycyrrhiza glabra roots

Noor Nihal Herbs Trading Company
House No.325/8 New No.421 “Yarab Manzil”,
1st Floor Mariyappa Layout, 1st Cross,
Pump House Road Gokul Extension,
Anekal -562 106, Bangalore.
2 Glabridin/ Glabridin extract Formulated in house
3 Licorice DPI Formulated in house
4 Glycyrrhizinic acid standard Natural remedies (Bangalore, India).

The abbreviations used in the invention are represented in Table 2 below.
Table 2: Legend of Abbreviations used.
Serial No. Term Full Form
1 CPCSEA Committee for the Purpose of Control and Supervision of Experiments on Animals
2 IAEC Institutional Animal Ethics Committee
3 Mg Milligram
4 Kg Kilogram
5 µL Microliter
6 FCA Freund Complete Adjuvant
7 SGOT Serum Glutamic Oxaloacetic Transaminase
8 SGPT Serum Glutamic Pyruvic Transaminase
9 ALP Alkaline phosphatase
10 CRP C-reactive protein
11 SOD Superoxide dismutase
12 GSH Glutathione
13 ESR Erythrocyte Sedimentation Rate

According to data from the Global RA Network, 2021, Arthritis is a common health problem in the global population, affecting more than 350 million people and a leading cause of disability. Arthritis causes more disability than any other condition, including heart disease, diabetes, and back or spine problems. Women have a greater tendency to develop it than men– and from babies to older people. Contrary to popular belief, arthritis is not a disease of the elderly; more than three in five people diagnosed with arthritis are under the age of 65. The burden of arthritis worldwide is expected to have significant consequences in terms of health care costs and loss of productivity by patients today, and over the next 30 years.

Piroxicam is a nonsteroidal anti-inflammatory drug of the oxicam class used to alleviate the symptoms of aching inflammatory conditions like Arthritis. Piroxicam acts by inhibiting the production of endogenous prostaglandins which are involved in the mediation of pain, stiffness, tenderness and swelling. People who take nonsteroidal anti-inflammatory medications (NSAIDs) may have a higher risk of having a heart attack or a stroke than people who do not take these medicines. These occurrences may happen without warning and may cause death. This risk may be higher for people who take NSAIDs for a long time.

Glabridin is a chemical compound that is found in the root extract of Licorice (Glycyrrhiza glabra). Glabridin is an isoflavane, a type of isoflavonoid. This compound is part of a larger family of plant-derived molecules, the natural phenols. Glabridin efficiently reduces platelet activation, so it might become therapeutic agent for thromboembolic disorders. It is used as a component in cosmetics and is listed in International Nomenclature of Cosmetic Ingredients (INCI). It is a yellowish-brown powder. It is insoluble in water, but soluble in organic solvents such as propylene glycol. The present invention studies Glabridin for its application in treatment of Arthritis along with two other drugs Favipiravir and Ivermectin.

Favipiravir (T-705) is a synthetic prodrug, first found while assessing the antiviral activity of chemical agents active against the influenza virus in the chemical library of Toyoma chemicals. A lead compound, A/PR/8/34, later described as T-1105, and its derivatives were found to have antiviral activities. Favipiravir is obtained by chemical alteration of the pyrazine moiety of T-1105.

Ivermectin is an anti-parasite medication used to cure parasitic diseases. It is FDA approved for use in humans to cure a variety of parasitic illnesses including parasitic worms, hookworm and whipworm. Ivermectin may also be used as a useful therapy for a wide range of other conditions like onchocerciasis, intestinal strongyloidiasis and onchocerciasis or river blindness.

The present invention discloses a pharmaceutical composition for improving conditions of Arthritis. The said composition is significantly efficient and effective for alleviating the symptoms of Arthritis.

The present invention provides an herbal pharmaceutical composition comprising of an active compound Glabridin which is effective in treating the disorder of Arthritis and exhibits minimum or no side effects.

Experiments have been conducted on the isolated compound Glabridin for its potential ability to be used for treating Arthritis. All the experimental work has been done following the protocols approved by Institutional Animal Ethics Committee (IAEC), Protocol number PCP/IAEC/2021-2022/2-72 constituted under guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). All the animals were euthanized after the completion of the study. The study was conducted using recommended/approved anesthetics and under the supervision of a veterinarian.

Extraction, isolation and purification of Glabridin from Glycyrrhiza glabra roots.
Extraction is the process of separating the pharmaceutically active mix of naturally active compounds contained inside plant materials or tissues using selective solvents using the standard techniques. It can be also defined as the treatment of the plant material with solvent, whereby the pharmaceutically active components are dissolved and most of the dormant matter remains undissolved. Therefore, the purpose of all extraction is to separate the soluble plant metabolites, leaving behind the unsolvable cellular excess.

Isolation and purification of the compound of interest using HPLC is the process of separating or extracting the target compound from other (possibly structurally related) compounds or contaminants. Each compound should have a characteristic peak under certain chromatographic conditions. Depending on what needs to be separated and how closely related the samples are, the chromatographer chooses the conditions, such as the proper mobile phase, flow rate, suitable detectors and columns to get an optimum separation.

Identification of compounds by HPLC is a critical part of any HPLC assay. The process of HPLC enables rapid and accurate identification of compounds in medicinal herbs. In order to distinguish any compound by HPLC, a detector must first be chosen. Once the detector is chosen and is set to optimal detection settings, a separation assay must be developed. The parameters of this assay are such that a clean peak of the known sample is observed from the chromatograph. The identifying peak is desired to have a reasonable retention time and is to be well separated from irrelevant peaks at the detection levels which the assay will be performed.

Extraction of Glabridin was done from Glycyrrhiza glabra roots of weight in the range of 100-500 gms, using a solvent at approximately 45-50°C for a duration of 3 hours in each cycle. Three cycles were repeated to get a good quantity of liquid extract. This liquid extract is then passed through a paper (Whatman paper) to concentrate. After extractions, all acetone extractions were pooled and concentrated on a rotary evaporator under vacuum at 45-50°C to get a dry powdered acetone extract of Glycyrrhiza glabra. This yields a significant high yield ofglabridrin. The extraction and isolation protocol from Glycyrrhiza glabra roots is illustrated as a flow chart as below.

Accordingly, in some embodiments, disclosure provides an efficient method for isolation of the active compound of interest, Glabridin. The fraction of the extract as obtained is subjected to Column chromatography for separating the active compound from the liquid extract. The extract is passed through a first column to obtain enriched Glabridin. It is further subjected to a second column to obtain a purified Glabridin. The purity of the active compound is ascertained using the HPLC method.

HPLC Method:
The Glabridin content in the powdered extracts recovered from the process was analyzed using an HPLC system consisting of a quaternary pump with a vacuum degasser, thermos tatted column compartment, autosampler, and UV detector. A reverse-phase column (Inertsil ODS) was used with a column temperature of 40 °C. The HPLC mobile phase Solution A or the buffer solution was prepared by dissolving Potassium dihydrogen phosphate in HPLC grade water. The solution was filtered through a membrane filter and degassed in a sonicator for 3 min; Solution B: Acetonitrile (100%). The mobile phase was run using gradient elution.
Mobile Phase A: Buffer solution as prepared.
Mobile Phase B: Acetonitrile.
Diluent: Methanol is used as a diluent.
Sample preparation
Glycyrrhiza glabra extract was weighed and transferred to a volumetric flask. Methanol was added and the contents were sonicated. The volume was then made up with methanol, passed through a filter and the resulting filtrate was used as a test solution. A stock solution of Glabridin standard was prepared in absolute methanol.
The purity of the compound Glabridin achieved through this process was found to be more than 95 % and a high yield of Glabridin was obtained of more than 7 % which is unprecedented. The Glabridin Standard Chromatogram and Isolated compound chromatogram are illustrated in Figures 1 and 2 respectively.
Therefore, the present invention provides a method of isolation of active compound Glabridin, in an efficient manner and hence said method is cost effective and scalable

Stability of Glabridin: The stability of Glabridin was studied for acid and alkaline hydrolysis. The pure Glabridin was exposed to acid and alkali hydrolysis with 1N HCl and 1N NaOH respectively for 1 hr at RT and the residual Glabridin was estimated using high performance liquid chromatography as per method given by Shrikant Kulkarni et al. 2021.

Study of efficacy of Glabridin on Hexokinase
In an embodiment of the invention, the inhibitory efficacy of Glabridin on hexokinase was studied using in-vitro hexokinase assay. The molecular mechanism of interaction between Glabridin and hexokinase enzyme was predicted using molecular docking techniques. The purified Glabridin as obtained, acts as hexokinase inhibitor. Said characteristic of Glabridin is being reported for the very first time by the present invention. Both in-vitro assays and molecular docking study have shown that Glabridin can be used as a potent natural inhibitor of hexokinase having better stability and higher bioactivity.

Molecular docking study:
Materials and methods: The in-vitro hexokinase assay method and the materials used for it are illustrated below.

Chemicals used
Hexokinase calorimetric assay kit (Product no: MAK91) and 2-Deoxy-D-glucose were purchased from Sigma Aldrich (Bangalore, India). Glabridin was isolated in-house with 95% purity. Licorice DPI was also formulated in-house. Glycyrrhizinic acid was procured from Natural remedies (Bangalore, India).

Hexokinase assay
The hexokinase (HK) assay was carried out using a hexokinase calorimetric assay kit as per instructions given by the manufacturer. NADH standard solution from HK assay kit was reconstituted with 400 µl water to generate 1.25 mM stock solution. From stock solution, 0, 2, 4, 6, 8, 10 µl aliquots were withdrawn into 96 well microtiter plate to achieve blank, 2.5, 5, 7.5, 10 and 12.5 nmol/well concentrations. One unit of HK is the amount of enzyme that will generate 1.0 µmole of NADH per minute at pH 8.0 at room temperature. To each 50 µL of sample solution containing inhibitor solution and HK enzyme was added followed by 50 µL of appropriate reaction mix (Table 3). For control samples, the same procedure was followed without addition of inhibitor solution. The samples were mixed in well by pipetting. The plate was incubated in the dark for 5 min at room temperature. After 5 min (Tinitial) incubation, initial absorbance [(A450)initial] was measured at 450 nm using ELISA plate reader (Erba LisaScan II). Plate was incubated for 60 min taking measurements every 10 min. Final measurement [(A450)final] and time of final reading (Tfinal) was used to calculate enzyme activity. Using these measurements, the change in measurement from Tinitial to Tfinal for the samples was calculated using the following formula.
?A450 = (A450) final – (A450) initial
The HK activity of a sample was determined by the following equation.

HK Activity =

Where,
B = Amount (nmole) of NADH generated between Tinitial and Tfinal.
Reaction Time = Tfinal – Tinitial (minutes)
V = Sample volume (mL) added to well
The relative hexokinase activity was calculated using the following formula.

Relative hexokinase activity =

Table 3: Composition of reaction mix
Sr. no Reagent Samples and standards Sample Blank
1 HK assay buffer 34µl 44 µl
2 HK enzyme mix 2µl 2 µl
3 HK Developer 2µl 2 µl
4 HK Coenzyme 2µl 2 µl
5 HK substrate 10 µl -

Results:
2 Deoxy D glucose (2-DG) is a glucose molecule in which the 2-hydroxyl group gets replaced by hydrogen. Due to this chemical replacement, 2DG is not able to enter glycolysis and contribute to ATP production. DG otherwise can be attached to various radioactive substrates and is being extensively used in various diagnostic tests and laboratory studies. It is considered to interrupt glycolysis and is a known hexokinase inhibitor.

The present study demonstrates HK inhibition activity of Glabridin, Licorice DPI and 2-DG by measuring relative HK activity. The calorimetric hexokinase assay is a coupled enzyme assay, based on conversion of glucose to glucose-6-phosphate by hexokinase, which is further oxidized by glucose-6-phosphate dehydrogenase to form NADH. The resulting NADH reduces a colorless probe resulting in a colorimetric (450 nm) product proportional to the hexokinase activity present.

In vitro hexokinase inhibition assay revealed that Glabridin at concentration of 10.8 mM inhibits 61.3 % hexokinase activity while the Licorice DPI had shown 60.65 % inhibitory activity. The 2-Dexoy-D-glucose, a known hexokinase inhibitor at final concentration of 45 mM had shown 37.25 % inhibitory activity. The reported half- maximal inhibitory concentration (IC50) for cystic epithelial cells treated with 2-Dexoy-D-glucoe is 30.9 mM as per Zhao et al which depends upon the assay conditions and the source of hexokinase enzyme used for the study. The glycyrrhizic acid, another important biomarker of Licorice, didn’t show any inhibitory activity against hexokinase. Hence, the inhibitory potential of Licorice DPI is due to the presence of Glabridin in the extract.

Licorice DPI has shown 60.65 % inhibitory activity against hexokinase which is significantly higher than 2- Deoxy-D-Glucose, thus Licorice DPI can be used as effective formulation for hexokinase inhibitory activity. The isolated Glabridin was found to be stable to acid and alkali hydrolysis with 1N HCl and 1N NaOH respectively for 1 hr at RT. Figure 3 illustrates the graph showing comparison of HK activity of 2-deoxy-2-glucose, Licorice DPI and pure Glabridin and Glycyrrhizin.

The ability of hexokinase inhibitors to inhibit glucose metabolism by inhibiting the first-rate limiting step of glycolysis can lead to nutrient and energy deprivation in cancer cells. This nutrient and energy deprivation can be used as an efficient tool to suppress cancer cell growth and survival. It was also reported that hexokinase inhibitors such as 2-Deoxy-D- glucose and metformin compete with glucose to bind hexokinase enzymes in the cells and inhibit the metabolism of tumor cells and can restrict the cell proliferation Zhao et al. In case of viral infection, the activity of hexokinases was increased by its interaction with virus proteins to fulfill the high energy requirement for metabolism, replication and protein synthesis.

Conclusion:
The study revealed that Glabridin and Licorice DPI could interfere in the glycolytic pathway by inhibiting hexokinase activity. The inhibition of HK will induce apoptosis and reduce viral cell replication.

Molecular docking of Glabridin with rheumatoid arthritis targets
Molecular Docking is an important component of computer-assisted drug discovery. It helps in predicting the intermolecular framework formed between a protein and ligand and outputs the appropriate binding between the molecules. The best conformation with the lowest docked energy is chosen from the docking search.

Extra Docking Information:
Essentially, the aim of molecular docking is to give a prediction of the ligand-receptor complex structure using computation methods. Docking can be achieved through two interrelated steps: first by sampling conformations of the ligand in the active site of the protein; then ranking these conformations via a scoring function. Ideally, sampling algorithms should be able to reproduce the experimental binding mode and the scoring function should also rank it highest among all generated conformations. Rheumatoid Arthritis (RA) is a chronic auto-immune degenerative disease that mainly affects synovial joints, progressing to functional disability. Caused due to dysfunctional intracellular signaling pathways (JAK/STAT, PI-3K/AKT/m-TOR, SAPK/MAPK) activated by pro-inflammatory cytokines.

AKT1(ASN53, LYS268, ASP292) leading site for cell cycle arrest and inhibiting inflammatory signals. Akt is a serine/threonine kinase that plays a central role in supporting proliferation and survival in a variety of cells. Phosphorylated Akt is overexpressed in the rheumatoid synovial tissue.

Mitogen activated protein kinase-1(MAPK1) - LYS54, GLU109 and MET108, are key binding sites that lead the structural change in glycine rich loop and protein kinase activation. MAPKshave been implicated as key regulators of pro-inflammatory cytokine (e.g. IL-1, IL-6, IL-12, IL-23 and TNF) production as well as playing crucial roles in signaling. Inhibition of p38 MAPK activity suppresses the TNF-a/IL-1-mediated induction of IL-6 by osteoblasts and chondrocytes that plays a role in osteoclast formation and bone resorption.
Cellular tumor antigen p53(TP53)-HIS115, ARG282, TYR126 responsible for DNA binding that activates p53 expression. Tumor suppressor’s function of p53 (i.e., its ability to induce apoptosis), when overexpressed early, may ameliorate the course of the disease.

MAP kinases are found in eukaryotes only, but they are fairly diverse and encountered in all animals, fungi and plants, and even in an array of unicellular eukaryotes. The mitogen-activated protein kinase (MAPK) family consists of both stresses activated (SAPK) and mitogen-activated (MAPK) protein kinases. They form a network of signal transduction cascades that mediate cellular responses to a diverse range of stimuli, including growth factors, chemical or osmotic stress, irradiation, bacterial infection and proinflammatory cytokines. Most MAPKs are activated by dual phosphorylation on a Thr-Xaa-Tyr motif by upstream kinases, referred to as MAPK kinases or MKKs. MKKs are, in turn, activated by MKK kinases (MKKKs), over 30 of which have been described. However, the details of how they are activated or which MKKK really activates which MKK in vivo is still poorly understood. MAPK cascades frequently function as multi-protein complexes in which the different components are assembled on a scaffold protein and/or by specific protein-protein docking sites, thereby increasing the speed and specificity of the cascade. Nearly all MAPKs phosphorylate their substrates on serine or threonine residues that precede a proline, but their specificity in vivo is further enhanced by the presence of distinct docking sites that facilitate interaction with substrates.

Tumor protein P53, also known as p53, cellular tumor antigen p53 (UniProt name), the Guardian of the Genome, phosphoprotein p53, tumor suppressor p53, antigen NY-CO-13, or transformation-related protein 53 (TRP53), is any isoform of a protein encoded by homologous genes in various organisms, such as TP53 (humans) and Trp53 (mice). This homolog (originally thought to be, and often spoken of as, a single protein) is crucial in multicellular vertebrates, where it prevents cancer formation. As such, p53 has been described as "the guardian of the genome" because of its role in conserving stability by preventing genome mutation. Hence TP53 is classified as a tumor suppressor gene. The name P53 was given in 1979 describing the apparent molecular mass; SDS-PAGE analysis indicates that it is a 53-kilodalton (kDa) protein. However, the actual mass of the full-length p53 protein (p53a) based on the sum of masses of the amino acid residues is only 43.7 kDa.

RAC (Rho family)-alpha serine/threonine-protein kinase is an enzyme that in humans is encoded by the AKT1 gene. This enzyme belongs to the AKT subfamily of serine/threonine kinases that contain SH2 (Src homology 2-like) protein domains. It is commonly referred to as PKB, or by both names as "Akt/PKB". The serine-threonine protein kinase AKT1 is catalytically inactive in serum-starved primary and immortalized fibroblasts. AKT1 and the related AKT2 are activated by platelet-derived growth factor. The activation is rapid and specific, and it is abrogated by mutations in the pleckstrin homology domain of AKT1. It was shown that the activation occurs through phosphatidylinositol 3-kinase. Mice lacking Akt1 display a 25% reduction in body mass, indicating that Akt1 is critical for transmitting growth-promoting signals, most likely via the IGF1 receptor. Mice lacking Akt1 are also resistant to cancer: They experience considerable delay in tumor growth initiated by the large T antigen or the Neu oncogene. A single-nucleotide polymorphism in this gene causes Proteus syndrome.

Comparative Docking Analysis Report
An embodiment of the disclosure examines the docking aspects of the active compound Glabridin with three proteins namely, Mitogen-activated protein kinase 1, Cellular Tumor Antigen p53 and RAC-alpha serine/threonine-protein kinase. The docking studies have been carried out using another drug Favipiravir which is a common antiviral drug to check if it can be repurposed for use in arthritis. The study also reports the docking analysis of the said three proteins with a popular treatment option available in the market known as Ivermectin. The details of the study and the results along with interpretation are presented in the following paragraphs. Figures 4-12 illustrate the docking of the three abovementioned proteins with the three ligands Glabridin, Favipiravir and Ivermectin.

Docking analysis
1WZY

Protein name: Mitogen-activated protein kinase 1, Organism(s): Homo sapiens, Sequence Length=368, Resolution: 2.50 Å, Uniport ID - P28482.

Ligands used for docking an analysis are as below.
Ligand name: Favipiravir, Molecular Formula: C5H4FN3O2, Molecular Weight: 157.1g/mol, PubChem CID: 492405
Ligand name: Glabridin, Molecular Formula: C20H20O4, Molecular Weight: 324.4g/mol, PubChem CID: 124052
Ligand name: Ivermectin, Molecular Formula: C48H74O14, Molecular Weight: 875.1g/mol, PubChem CID: 6321424.The details of the study are illustrated in tables 4-6 below.

Table 4: Ligand information:

Protein Name Ligand Name Binding energy
(kcal/mol) No. H Bonds Interacting residue Final Intermolecular Energy (kcal/mol) vdW + Hbond + desolv Energy (kcal/mol) Electrostatic
Energy (kcal/mol) Torsional Free Energy (kcal/mol)
1WZY Favipiravir

-4.01 02
(H1=2.11Å),
(H2=2.71Å). LEU:267(H1),
LEU:265(H3),
HIS:269,
LEU:252,
PRO:247. -3.82 -3.47 -0.35 +0.30
Glabridin

-6.65 01
(H1=2.11Å).
LEU:278(H1),
ILE:227,
PHE:228,
PRO:229,
TRP:192,
HIS:239,
LYS:231. -7.58 -7.37 -0.20 +0.89
Ivermectin

-8.40 01
(H1=2.92Å).
ASP:332(H1),
LEU:346,
MET:333,
LEU:338. -7.04 -6.33 -0.71 +3.28

The Grid Data information is as given below.
Grid Point Spacing = 1.000 Angstroms,
Even Number of User-specified Grid Points = 56 x-points
40 y-points
60 z-points
Coordinates of Central Grid Point of Maps= (-2.015, 3.569, 37.145)
Minimum coordinates in grid = (-30.015, -16.431, 7.145)
Maximum coordinates in grid = (25.985, 23.569, 67.145)

Table 5: Lipinski’s Rule information:
Sr. No. Name of Co-former Mol
Weight (g/mol) XLogP3 Hydrogen Bond Donor Hydrogen Bond Acceptor Rotatable Bond
1 Favipiravir 157.10 -0.6 2 4 1
2 Glabridin 324.4 3.9 2 4 1
3 Ivermectin 875.1 4.1 3 14 8

Table 6: Structure of the Ligands used:
Sr. No. Ligand Name 2D Structure
1 Favipiravir

2 Glabridin

3 Ivermectin

2BIN
Protein name: Cellular Tumor Antigen P53, Organism(s): Homo sapiens, Sequence Length=219, Resolution: 1.90 Å, Uniport ID - P04637.
The Ligand information is presented in Table 7.

Table 7: Ligand information -
Protein Name Ligand Name Binding energy(kcal/mol) No. H Bonds Interacting residue Final Intermolecular Energy (kcal/mol) vdW + Hbond + desolv Energy (kcal/mol) Electrostatic Energy (kcal/mol) Torsional Free Energy (kcal/mol)
2BIN Favipiravir -3.85 02
(H1=2.14Å),
(H2=2.78Å). PRO151(H1), THR:155(H2), PRO:222, PRO:153. -4.01 -3.91 -0.10 +0.30
Glabridin -5.63 02
(H1=1.99Å),
(H2=3.34Å). PHE:113(H1), ARG:282(H2),ARG:110,TRP:146. -6.53 -6.56 +0.04 +0.89
Ivermectin -7.71 0 ARG:110, TRP:146. -6.02 -6.01 -0.01 +3.28

The Grid Data information is as given below.
Grid Point Spacing =1.000 Angstroms,
Even Number of User-specified Grid Points = 40 x-points
40 y-points
40 z-points

Coordinates of Central Grid Point of Maps= (124.731, -29.274, -263.607)
Minimum coordinates in grid = (104.731, -49.274, -283.607)
Maximum coordinates in grid (144.731, -9.274, -243.607)

6S9X
Protein name: RAC-alpha serine/threonine-protein kinase, Organism(s): Homo sapiens, Sequence Length=446, Resolution: 2.60 Å, Uniport ID - P31749
The Ligand information is presented in Table 8.

Table 8: Ligand information
Protein Name Ligand Name Binding energy(kcal/mol) No. H Bonds Interacting residue Final Intermolecular Energy (kcal/mol) vdW + Hbond + desolv Energy (kcal/mol) Electrostatic
Energy (kcal/mol) Torsional Free Energy (kcal/mol)
6S9X Favipiravir -4.33 04
(H1=1.98),
(H2=2.31),
(H3=2.26Å),
(H4=2.27Å). ASP:325(H1) GLY:327(H2), ALA:329(H3),GLY:394(H4),LYS:389,PRO:388. -4.21 -4.17 -0.04 +0.30
Glabridin -6.50 01
(H1=1.85Å).
LYS:268(H1),
ASN:53,
VAL:270,
TRP:80 -7.46 -7.35 -0.11 +0.89
Ivermectin -9.52 01
(H1=3.14Å).
TYR:350(H1), ARG:346,
ARG:243,
PHE:442,
PHE:236. -7.47 -7.36 -0.11 +3.28

The Grid Data information is as given below.
Grid Point Spacing =1.000 Angstroms,
Even Number of User-specified Grid Points = 62 x-points
44 y-points
54 z-points
Coordinates of Central Grid Point of Maps= (3.881, 2.131, 9.134)
Minimum coordinates in grid = (-27.119, -19.869, -17.866)
Maximum coordinates in grid = (34.881, 24.131, 36.134)

Animal studies
In an embodiment the potency of Glabridin, Favipiravir and Ivermectin for treatment of Rheumatoid arthritis has been tested on arthritis induced animal wherein it was observed that Glabridin has potent anti-arthritic activity. This was then followed by animal studies to check the efficacy of pharmaceutical formulation containing active compound Glabridin of two different dosages.

Studies to check the effect of Glabridin in comparison to Favipiravir and Ivermectin
The purpose of the study was to check the effect of Glabridin, Favipiravir and Ivermectin in Freund’s Complete Adjuvant induced chronic inflammation in female wistar rats. It is known that abnormal glycolytic metabolism contributes to joint inflammation and destruction in rheumatoid arthritis (RA). Hence the use of Glabridin, Favipiravir and IVM in alleviating arthritis was explored by animal models. Arthritis was induced in rats by subplantar injection of Freund’s Complete adjuvant.

Arthritis animal model and the method of studies:
Female wistar rats (180-250 g) were divided into 6 groups of 6 animals each. The groups were made as below.
Group 1: Vehicle control,
Group 2: FCA control,
Group 3: Piroxicam (Piroxicam is a nonsteroidal anti-inflammatory drug of the oxicam class used to relieve the symptoms of painful inflammatory conditions like arthritis) treated.
Group 4-6: SAVA 14 A (Glabridin), 14 B (Favipiravir), 14 C (Ivermectin).
The experimental chronic inflammation was induced to all groups except group 1 by the dose of FCA (0.1 ml FCA consists of complete fraction of Mycobacterium butyricum being suspended in heavy paraffin oil) by subplantar injection in the left paw of rat on 0 day. An additional challenge of 0.1 ml FCA was given on day 8.
A total of 12 days of time duration were given to produce chronic inflammation. Everyday animals were inspected by examining the affected paw, body weight and the animal’s general status.

The selected doses of SAVA 14 A, 14 B, 14 C or Piroxicam were administered from 12th day till 28 days daily as injections by subcutaneous route after induction of inflammation. Piroxicam was given by i.p route as injections at (7.5mg/dose/rat), Glabridin was given as 100 µl injection (2mg/dose/rat), Favipiravir as a clear solution with arginine as 200 µl injection (2mg/dose/rat) and Ivermectin as a clear solution as 200 µl injection in PG (2mg/dose/rat).

The primary lesions were determined by measuring paw volume on the day 0, 4, 8, 12, 16, 20, 24 and 28th day (using Plethysmometer) and pain threshold were measured on day 0, 12 and 28 (using Randall-Sellitto apparatus).

The following parameters were evaluated on various days:
Body weight (g): Chronic inflammation affects the body weight. Body weight (g) was measured on day 0, day 12 and day 28.
Paw volumes were measured on day 0, 4, 8, 12, 16, 20, 24 and 28.
ESR reflects the chronicity of the disease were measured on day 28 (end of treatment).
Serum C-reactive protein level (mg/l): C-reactive protein (CRP) concentrations are useful plasma protein measure that correlates with disease severity and radiographic progression in chronic inflammation. It was measured at the end of the experiment. The serum SGOT, SGPT, ALP, liver SOD and liver GSH levels were also determined at the end of experimentation.
Histopathology: The histopathology of joint tissue was performed on the day 28 after sacrificing the animals.

Results
Effect of SAVA 14A, SAVA 14B and SAVA 14C on body weight (g) of animals
There was no significant difference observed in the body weight of all the rats in FCA, piroxicam, SAVA 14A, SAVA 14B and SAVA 14C treated groups on day 0 to day 28 during the treatment period (Table 9).

Effect of SAVA 14A, SAVA 14B and SAVA 14C on Paw volume (ml)
The progression of FCA induced arthritis was evaluated by measuring the paw volume using a plethysmometer on days 1, 4, 8, 12, 16, 20, 24 and 28. The change in paw volume was calculated as the difference between the final (28 days) and initial (0 days) paw volume. In FCA treated rats, there was a significant (p<0.001) increase in paw volume seen from day 4 to 28 when compared to VC rats (Table 9). Treatment of rats with PXC, SAVA 14A and SAVA 14B exhibited significant (p<0.05; p<0.01; p<0.001) decrease in the paw volume from day 16 to day 28 when compared to FCA treated rats. However, the rats treated with SAVA 14C did not show any significant decrement in the paw volume when compared to FCA rats.

Effect of SAVA 14A, SAVA 14B and SAVA 14C on Pain threshold (gf/N/Lbf)
The pain threshold was significantly (p<0.05; p<0.001) increased in FCA treated rats when compared to VC rats from day 1 to day 28 during the study period. However, no significant decrement in the pain threshold was observed in the rats treated with PXC, SAVA 14A, SAVA 14B and SAVA 14C as compared to FCA treated rats from day 1 to day 28(Table 9).

Effect of SAVA 14A, SAVA 14B and SAVA 14C on SGPT (U/L)
On day 28 there was a significant difference in SGPT (U/L) level in FCA treated rats when compared to VC rats. Further on treatment of rats with PXC, SAVA 14A, SAVA 14B and SAVA 14C, did not cause any significant decrease in the SGPT levels when compared with FCA treated rats on day 28 (Table 10).

Effect of SAVA 14A, SAVA 14B and SAVA 14C on SGOT (U/L)
There was a significant (p<0.001) difference observed in SGOT (U/L) levels in rats treated with FCA when compared with VC rats on day 28. Further on treatment of rats with PXC, SAVA 14A, SAVA 14B and SAVA 14C for 28 days exhibited significant (p<0.001) decrease in the SGOT levels in SAVA 14A, SAVA 14B and SAVA 14C treated animals when compared with FCA treated rats on day 28 (Table 10).

Effect of SAVA 14A, SAVA 14B and SAVA 14C on ALP (IU/L)
There was a significant (p<0.001) difference observed in ALP (IU/L) levels in rats treated with FCA when compared with VC rats on day 28. Further on treatment of rats with SAVA 14A and SAVA 14B for 28 days exhibited significant (p<0.001) decrease in the ALP levels in SAVA 14A and SAVA 14B treated animals when compared with FCA treated rats. However, rats treated with SAVA 14C did not show any significant decrease in the ALP levels when compared with FCA treated rats on 28 days (Table 10).

Effect of SAVA 14A, SAVA 14B and SAVA 14C on CRP (mg/L)
There was a significant increase in the CRP (mg/L) levels of FCA control group of animals when compared to vehicle control. Treatment with SAVA 14A and SAVA 14B caused a significant (p<0.01 and p<0.001, respectively) decrease in the CRP level as compared to FCA treated rats on day 28 (Table 10). However, SAVA 14C treatment did not cause a change in CRP level when compared to FCA treated rats.

Effect of SAVA 14A, SAVA 14B and SAVA 14C on ESR (mm/h)
There was a significant (p<0.001) increase in the ESR (mm/h) levels observed in FCA rats when compared to VC rats. Treatment of rats with PXC, SAVA 14A, SAVA 14B and SAVA 14C showed significant (p<0.001) decrement in ESR levels as compared to FCA treated rats on day 28 (Table 10).

Effect of SAVA 14A, SAVA 14B and SAVA 14C on SOD (U/mg)
There was significant (p<0.001) decrease observed in SOD (U/mg) levels in rats FCA treated when compared to VC rats on day 28. Treatment of rats with PXC, SAVA 14A, SAVA 14B and SAVA 14C for 28 days exhibited significant (***p<0.001) increase in the SOD levels when compared to FCA rats on day 28 (Table 10).

Effect of SAVA 14A, SAVA 14B and SAVA 14C on GSH (µmol/g)
There was significant (p<0.001) decrease observed in GSH (µmol/g) levels in rats FCA treated when compared to VC rats on 28 days. Further on treatment of rats with PXC, SAVA 14A and SAVA 14B for 28 days exhibited significant ((p<0.001) increase in the GSH levels when compared with FCA rats on day 28 (Table 10). However, there was no significant change in the SAVA 14C treated animals.

Conclusion:
In the present study, SAVA 14 A (Glabridin) was found to be very effective in treating arthritis as evident by decrease in paw volume, pain threshold and biochemical parameters when compared to FCA (arthritic) control.
SAVA 14B Favipiravir (200 µl/animal, s.c) was found to be moderately effective.
However, SAVA 14 C (IVM) did not have any effect in inflammation, pain or the biochemical parameters.

Table 9: Effect of SAVA 14 on Body weight, Paw volume and Pain threshold in Freund’s Complete Adjuvant induced Arthritis
Parameters Day VC FCA Piroxicam (positive control) Glabridin
(100µl) Favipiravir
(200µl) Ivermectin (200µl)
Body Weight (g) 1 184.50 ±
1.89 204.67±
4.11 198.83±
4.80 198.83±
7.30 208.00±
3.71 200.50±
7.55
28 206.50 ±
6.48 184.17±
4.59 182.83±
5.08 180.67±
7.10 185.83±
5.90 180.00±
9.31
Paw Volume
(ml) 1 1.75±
0.09 1.65±
0.08 1.61±
0.09 1.55 ±
0.02 1.56 ±
0.02 1.41±
0.08
28 1.91±
0.03 3.76±
0.08### 2.28 ±
0.09*** 2.90 ±
0.02*** 3.32 ±
0.04*** 3.49 ±
0.06
Paw Withdrawal threshold (g) 1 346.67 ±
32.44 378.67 ±
62.06 352.00 ±
11.68 346.67 ±
12.84 352.00 ±
11.68 346.67 ±
9.83
28 320.00 ±
16.52 122.67 ±
5.33### 266.67 ±
13.49*** 240.00 ±
23.00*** 277.33 ±
6.75*** 106.67 ±
6.75

VC: Vehicle Control; FCA: Freund’s Complete Adjuvant; PXC: Piroxicam
Values are represented as mean ± SEM, n = 6
Two-way ANOVA followed by Dunnet’s multiple comparison test.
#p<0.05; ###p<0.001 when compared to VC; *p<0.01; **p<0.01; ***p<0.001 when compared to FCA control

Table 10: Effect of SAVA 14 on Biochemical parameters in Freund’s Complete Adjuvant induced Arthritis in Female rats
Parameters VC FCA PXC Glabridin
(100µl) Favipiravir
(200µl) Ivermectin (200µl)
SGOT (U/L) 53.17±
1.40 60.83±
1.99# 48.17±
1.25 51.83±
4.26 46.83±
2.18 52.17±
1.96
SGPT (U/L) 127.50±
7.01 223.80±
9.99### 166.80±
8.97** 119.50±
8.46*** 151.40±
5.81*** 147.10±
12.78***
ALP (IU/L) 152.10±
7.86 299.50±
33.33### 147.10±
4.88*** 184.3.00±
28.15*** 134.80±
7.92*** 262.00±
16.95
C-RP (mg/L) 13.99±
0.85 45.84±
1.67### 31.81±
0.88*** 38.70±
2.46** 31.92±
0.64*** 43.24±
1.21
ESR (mm/hr)
2.4 ±
0.02 9.13 ±
0.07### 3.51 ± 0.02*** 4.29 ±
0.03*** 3.90 ±
0.02*** 3.20 ± 0.03***
SOD (U/mg) 13.05 ±
0.28 8.96±
0.20### 10.17 ±
0.14*** 10.12 ±
0.06*** 9.48 ±
0.14*** 8.58±
0.21
GSH (µmol/g) 12.96 ±
0.44 9.10 ±
0.18### 12.57 ±
0.15*** 13.58 ±
0.32*** 13.21 ±
0.40*** 8.83 ±
0.43

VC: Vehicle Control; FCA: Freund’s Complete Adjuvant; PXC: Piroxicam
Values are represented as mean ± SEM, n = 6
One way ANOVA followed by Dunnet’s multiple comparison test.
#p<0.05; ##p<0.01; ###p<0.001; when compared to Vehicle Control
*p<0.01, **p<0.01; ***p<0.001 when compared to FCA control

Studies to check the effect of Glabridin with different dosages
In another embodiment of the invention, study of the effect of the Licorice extract (SAVA 18) with Glabridin as an active compound, of two different dosages through oral route of administration, on Freund’s Complete Adjuvant induced chronic inflammation in female wistar rats has been presented.

Objectives of the study:
a. To induce arthritis by subplantar injection of Freund’s Complete adjuvant (FCA).
b. To study the effect of SAVA 18 on inflammatory parameters.
c. To evaluate the biochemical parameters on SAVA 18 in Freund’s Complete adjuvant induced arthritis.

Materials and Methods used in the study:
Materials:
SGPT (ALAT), SGOT (ASAT), alkaline phosphate (ALP) and other chemicals and reagents used for the study were of analytical grade from approved vendors. All the experimental protocols were approved (Protocol approval number: PCP/IAEC/2021-2022/2-72) by Institutional Animal Ethics Committee of Poona College of Pharmacy, registered with CPCSEA with registration number 1703/PO/Re/S/01/CPCSEA dated 17/06/2016. 9-10 weeks old Female Wistar rats were procured. The animals were housed in polypropylene cages under maintained environment with temperature 25±1°C, relative humidity 45-55% and 12hr light: 12hr dark cycle. The animals had free access to feed pellets (VRK Nutritional Solutions, Pune) and water ad libitum.

Methods:
Female wistar rats (180-250 g) were divided into 5 groups of 6 animals each. Group 1: Vehicle control, Group 2: FCA control, Group 3: Piroxicam treated; Group 4: SAVA 18A; and Group 5: 18B treated groups.

The experimental chronic inflammation was induced to all groups except group 1 by the dose of FCA (0.1 ml FCA consists of complete fraction of Mycobacterium butyricum being suspended in heavy paraffin oil) by subplantar injection in the left paw of rat on 0 day. An additional challenge of 0.1 ml FCA was given on day 8. A total of 12 days of time duration was given to produce chronic inflammation. Everyday animals were inspected by examining the affected paw, body weight and the animal’s general status.

The selected doses of SAVA 18A and SAVA 18B are the extracts of Licorice. The selected doses of SAVA 18A (30 mg/animal), SAVA18B (60 mg/ animal), or Piroxicam (30 mg/Kg as 0.3 ml) were administered from 12th day till 28 days daily as oral administration. The primary lesion was determined by measuring paw volume on the day 0, 4, 8, 12, 16, 20, 24 and 28th day (using Plethysmometer) and pain threshold were measured on day 0, 12 and 28 (using Randall-Sellitto apparatus).

The following parameters were evaluated on various days:
Body weight (g): Chronic inflammation affects body weight. Body weight (g) was measured on day 0, day 12 and day 28.
Paw volumes were measured on day 0, 8, 12 and 28.
ESR reflects the chronicity of the disease were measured on day 28 (end of treatment)
Serum C-reactive protein level (mg/l): C-reactive protein (CRP) concentrations are useful plasma protein measure that correlates with disease severity and radiographic progression in chronic inflammation. It was measured at the end of the experiment. The serum SGOT, SGPT, ALP, liver SOD and liver GSH levels were also determined at the end of experimentation.
Histopathology: The histopathology of joint tissue was performed on the day 28 after sacrificing the animals.

Statistical Analysis: The data was expressed as mean ± SEM. The data was analyzed by Two-way ANOVA followed by Bonferroni’s test and day 28 biochemical parameters were analyzed by one way ANOVA followed by Dunnet’s ‘t’ test.
Results
Effect of SAVA 18A and SAVA 18B on body weight (g) of animals
Administration of animals with FCA significantly (p<0.001) reduced the body weight when compared with VC rats. The rats treated with piroxicam, SAVA 18A and SAVA 18B on day 28 exhibited significant (p<0.01) increase in the body weight when compared to FCA treated rats (Figure 13 and Table 11). However, there was no significant difference in body weight between SAVA 18A and SAVA 18 B group of animals.

Effect of SAVA 18A and SAVA 18B on Paw volume (ml)

There was a significant (p<0.001) increase in the ESR (mm/h) levels observed in FCA control group when compared to VC rats. Treatment of animals with PXC, SAVA 18A and SAVA 18B showed significant (p<0.001) decrease in ESR levels as compared to FCA control rats (Figure 14 and Table 11). There was a significant (p<0.001) decrease in ESR in SAVA 18 B treated group when compared to SAVA 18A treated group. ESR and CRP are reported to have increased in inflammation. The study confirms the effect of SAVA 18 in attenuation of inflammation. SAVA 18 B is more effective than SAVA 18 A in decreasing the inflammatory markers.

Effect of SAVA 18A and SAVA 18B on Pain threshold (gf/N/Lbf)
There was a significant (p<0.001) decrease in pain threshold in FCA induced rats when compared to VC rats. This indicated increased hyperalgesic pain in FCA induced animals. There was a significant (p<0.001) increase in the pain threshold observed in the rats treated with PXC, SAVA 18A and SAVA 18B as compared to FCA treated rats on day 28 (Figure 15 and Table 11). Treatment with SAVA 18B caused a significant (p<0.05) improvement in pain threshold when compared to SAVA 18A group. The parameters paw volume and pain threshold confirm that the SAVA 18B treatment has markedly significant anti-inflammatory effect and has a moderately significant analgesic effect.

Effect of SAVA 18A and SAVA 18B on CRP (mg/L)
There was a significant (p<0.001) increase in the CRP (mg/L) levels of FCA control group of animals when compared to VC rats. Treatment with SAVA 18A and SAVA 18B caused a significant (p<0.001) decrease in the CRP level as compared to FCA treated rats (Figure 16 and Table 12). C-reactive protein is a marker of inflammation. A significant decrease in the levels of CRP upon treatment with SAVA 18 confirms the role of SAVA 18 in attenuating inflammation. There was a 33 % decrease in CRP level upon treatment with SAVA 18A while, treatment with SAVA 18B caused a 41 % decrease in CRP level. However, the decrease between SAVA 18A and SAVA 18B was not significant.

Effect of SAVA 18A and SAVA 18B on ESR (mm/h)
There was a significant (p<0.001) increase in the ESR (mm/h) levels observed in FCA control group when compared to VC rats. Treatment of animals with PXC, SAVA 18A and SAVA 18B showed significant (p<0.001) decrease in ESR levels as compared to FCA control rats (Figure 17 and Table 12). There was a significant (p<0.001) decrease in ESR in SAVA 18 B treated group when compared to SAVA 18A treated group. ESR and CRP are reported to have increased in inflammation. The study confirms the effect of SAVA 18 in attenuation of inflammation. SAVA 18 B is more effective than SAVA 18 A in decreasing the inflammatory markers.

Effect of SAVA 18A and SAVA 18B on SGPT (U/L)
On day 28 there was a significant (p<0.001) increase in SGPT (U/L) level in FCA induced rats when compared to VC rats. Treatment of rats with PXC, SAVA 18A and SAVA 18B displayed significant (p<0.001) decrease in the SGPT levels when compared to FCA treated rats (Figure 18 and Table 12). However, there was no significant change in SGPT level between SAVA 18A and SAVA 18B.

Effect of SAVA 18A and SAVA 18B on SGOT (U/L)
There was a significant (p<0.001) increase observed in SGOT (U/L) levels in rats induced with FCA when compared to VC rats. Treatment with PXC, SAVA 18A and SAVA 18B for 28 days exhibited significant (p<0.01; p<0.01; p<0.001, respectively) decrease in the SGOT levels in SAVA 18A and SAVA 18B treated animals when compared with FCA treated rats on day 28 (Figure 19 and Table 12). However, there was no significant change in SGPT level between SAVA 18A and SAVA 18B.

Effect of SAVA 18A and SAVA 18B on ALP (IU/L)
There was a significant (p<0.001) difference observed in ALP (IU/L) levels in rats induced with FCA when compared to VC rats. Treatment with SAVA 18A and SAVA 18B for 28 days caused significant (p<0.001) decrease in the ALP levels in SAVA 18A and SAVA 18B treated animals when compared with FCA treated rats. (Figure 20 and Table 12). There was a significant (p<0.01) decrease in ALP level in SAVA 18B when compared to SAVA 18A. Increased concentration of serum alkaline phosphatase (ALP) is a common feature in rheumatoid arthritis due to increased bone resorption. The bone specific isoform of the enzyme serum alkaline phosphatase is a glycoprotein found on the surface of osteoblasts and reflects the activity of these cells in bone metabolism. In the present study, decrease in ALP by SAVA 18 confirmed the role of SAVA 18 in decreasing the bone resorption. SAVA 18B was more effective compared to SAVA 18A in decreasing the ALP.

Effect of SAVA 18A and SAVA 18B on SOD (U/mg)
There was a significant (p<0.001) decrease observed in SOD levels in FCA induced rats when compared to VC rats. Treatment of rats with PXC, SAVA 18A and SAVA 18B for 28 days exhibited significant (p<0.001) increase in the SOD levels when compared to FCA rats on day 28 (Figure 21 and Table 12).

Effect of SAVA 18A and SAVA 18B on GSH (µmol/g)
There was a significant (p<0.001) decrease observed in GSH (µmol/g) levels in FCA induced rats when compared to VC rats on 28 days. Further on treatment of rats with PXC, SAVA 18A and SAVA 18B for 28 days exhibited significant (p<0.001) increase in the GSH levels when compared with FCA rats on day 28 (Figure 22 and Table 12). Induction of arthritis causes an increase in reactive oxidative species and in turn, a decrease in antioxidant markers like SOD and GSH. There is a severe decline in glutathione defense system in arthritis. The results of SOD and GSH confirm that SAVA 18 plays a role in attenuation of inflammation and arthritis which lead to increase in the oxidative defense mechanism.

Conclusion
The Glycyrrhiza glabra extract has active phytochemical constituents of Glabridin, Liquiritigenin, Isoliquiritigenin which appear to contribute to the anti-arthritic effect. However, the content distribution of the Glabra extract is Glabridin 7.2% compared to 0.091 % of Liquiritigenin, 0.078 % of Isoliquiritigenin and Isoliquiritin which was not detected. Hence, it is asserted that the anti-arthritic potential of the Glycyrrhiza glabra extract is due to the presence of Glabridin in the extract.
In the present study, SAVA 18B (60 mg/animal, oral) was found to be very effective in treating the arthritis as evident by decrease in paw volume, pain threshold and biochemical parameters when compared to FCA (arthritic) control.
SAVA 18A (30 mg/ animal, oral) was found to be moderately effective in the treatment of FCA induced arthritis in rats.
There was a significant change between SAVA 18B and SAVA 18A in inflammation, hyperalgesia and ALP levels, which confirms that the high dose in the range of 60 mg/animal to 70 mg/animal has caused a marked improvement in arthritis compared to the low dose. This dose translates to almost 3 g of licorice/65 kg human/day. This is well within the permitted dose of 5-10 g of the licorice powder as per FSSAI list and as per Ayurvedic pharmacopoeia (2-4 g powder /day).

Table 11: Effect of SAVA 18 on Body weight, Paw volume and Pain threshold in Freund’s Complete Adjuvant induced Arthritis.
Parameters Day VC FCA PXC
(30 mg/Kg) SAVA 18A
(30 mg/ animal) SAVA 18B
(60 mg/ animal)

Body Weight (g) 1 191.50 ± 3.02 192.33 ± 3.46 191.00 ± 4.96 185.82 ± 8.06 195.32 ±
5.54
12 210.32 ± 3.84 195.16 ± 3.22 200.16 ± 4.68 191.32 ± 7.08 202.00 ± 5.78
28 226.32 ± 3.32 198.33 ± 2.48### 220.32 ± 3.44** 218.32 ± 3.28** 220.82 ± 1.78**

Paw Volume
(ml) 1 1.72 ± 0.09 1.64 ±
0.06 1.52 ±
0.04 1.49 ±
0.04 1.49 ±
0.04
8 1.92 ± 0.04 3.52 ±
0.08### 3.64 ± 0.06### 3.44 ± 0.02### 3.44 ± 0.04###
12 1.98 ± 0.04 3.79 ±
0.04### 3.76 ± 0.03### 3.75 ± 0.04### 3.68 ± 0.04###
28 2.05 ± 0.02 3.88 ±
0.06### 2.32 ±
0.04*** 3.16 ±
0.04*** 2.40 ± 0.04***

Paw Withdrawal threshold (g)
1 378.66 ± 26.66 314.66 ± 34.48 378.66 ± 15.26 368.00 ± 18.00 336.00 ± 37.64
8 409.32 ± 19.82 90.66 ± 18.32### 77.32 ± 11.24### 72.00 ± 12.90### 93.32 ± 17.24###
12 378.66 ± 26.66 72.00 ± 8.00### 69.32 ± 12.16### 69.32 ± 8.92### 53.32 ± 3.36###
28 376.50 ± 9.92 80.00 ± 13.06### 252.00 ± 9.12*** 208.00 ± 7.16*** 277.32 ± 5.32***

VC: Vehicle Control; FCA: Freund’s Complete Adjuvant; PXC: Piroxicam
Values are represented as mean ± SEM, n = 6
Two-way ANOVA followed by Bonferroni’s multiple comparison test.
###p<0.001 when compared to VC; ***p<0.001 when compared to FCA control

Table 12: Effect of SAVA 18 on Biochemical parameters in Freund’s Complete Adjuvant induced Arthritis in Female rats.
Parameters VC FCA PXC
(30 mg/Kg) SAVA 18A
(30 mg/ animal) SAVA 18B
(60 mg/ animal)
C-RP (mg/L) 15.21±
0.72 49.60 ±
1.98### 30.51±
0.80*** 33.03 ±
1.02*** 29.14 ±
0.30***
ESR (mm/hr)
1.76 ±
0.14 9.62 ±
0.36### 4.46 ± 0.08*** 5.30 ±
0.12*** 3.62 ±
0.18***
SGPT (U/L) 39.50±
1.38 54.82 ±
2.84### 41.66 ±
1.04*** 45.50±
1.40** 43.66 ±
1.48***
SGOT (U/L) 107.90 ±
3.38 198.50 ±
9.08### 163.90 ±
7.32** 173.40 ±
5.84* 156.60 ±
3.42***
ALP (IU/L) 160.50±
7.18 294.50±
15.90### 147.70±
3.38*** 190.50 ±
7.18*** 143.30±
4.22***
SOD (U/mg) 12.70 ±
0.70 7.40 ±
0.36### 9.84 ±
0.22*** 9.32±
0.12*** 9.68 ±
0.10***
GSH (µmol/g) 13.30 ±
0.30 8.96 ±
0.14### 11.86 ±
0.16*** 12.22 ±
0.16*** 12.86 ±
0.18***

VC: Vehicle Control; FCA: Freund’s Complete Adjuvant; PXC: Piroxicam
Values are represented as mean ± SEM, n = 6
One way ANOVA followed by Dunnet’s multiple comparison test.
###p<0.001; when compared to Vehicle Control
*p<0.01, **p<0.01; ***p<0.001 when compared to FCA control

Process of preparation of pharmaceutical composition
In an embodiment, the present invention relates to the process of preparation of a pharmaceutical composition with active compound Glabridin for improving conditions in Arthritis.
In a preferred embodiment the formulation comprises of extracts of Glycyrrhiza glabra with active compound, Glabridin, a biological marker of Licorice (Glycyrrhiza glabra), along with other excipients.

In another embodiment the formulation comprises of either extracts of Glycyrrhiza glabra comprising of Glabridin or isolated purified Glabridin as such, mixed along with other excipients for improving conditions of Arthritis.

In an embodiment, the formulation also comprises excipients to include a microcrystalline cellulose, an anti-caking agent, an additive for lubrication and purified water, further also comprises ethanol as solvent. The oral form can be in capsule tablet forms to include Vcap HPMC size “0” capsules clear cap/clear body.

In another embodiment, the present invention relates to a process of preparing a pharmaceutical composition for oral delivery of Glycyrrhiza glabra extract or isolated purified Glabridin, which is prepared using following steps:
Obtaining Glabridin as an acetone extract or isolated purified Glabridin from the plant source.
Mixing Glabridin extract or isolated purified Glabridin and microcrystalline cellulose.
Wet granulation of the mixture in previous step is carried out by adding purified water.
Blending is performed by adding an anti-caking agent to the above mixture.
Addition of additive for lubrication to the mixture.
The above mixture is then filled in the capsule.

The anti-caking agent is selected from a group comprising colloidal silicon dioxide (Aerosil 200), magnesium silicate, ammonium chloride, dioxins, non-colloidal MCC etc. The additive for lubrication is selected from a group comprising magnesium stearate stearic acid, potassium stearate, lithium stearate etc.

Pharmaceutical composition
As per an optimized embodiment the portions of the ingredients used for formulation is as tabulated below:

Table 13: Formulation process and ingredients of pharmaceutical composition
Sr. no Ingredients Mg/cap
1 Glycyrrhiza glabra extract or isolated purified Glabridin 1.00- 400.00
2 Microcrystalline cellulose (Avicel PH 101) 1.00-300.00
Wet granulation
3 Purified water q. s
Blending
4 Colloidal silicon dioxide (Aerosil 200) 0.1 to 10.00
Lubrication
5 Magnesium stearate 0.1 to 10.00
Total 100.00 - 600.00
6 Vcap HPMC size “0” capsules clear cap/clear body 1

The blend was found to be free flowing.

EXAMPLES
For a better understanding of this invention, the following examples are presented. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any manner.

Glabridin Extraction Trial:
For extraction of Glabridin, 300 grams of Glycyrrhiza glabra roots were taken and treated with 5 volumes of Acetone as solvent at approximately 50° C. Each cycle of extraction took 3 hrs. Three cycles were repeated to get a good quantity of liquid extract. This liquid extract is then passed through Whatman paper to get a concentrate. The concentrate is dried using a Rotary evaporator at a temperature of 40-45° C. This yields a significantly high yield of 15 grams of Licorice extract which amounts to 5 %.

15 grams of acetone extract is used for Column chromatography for further isolation and purification. The extract is enriched in this first column to obtain enriched Glabridin of 2.2 grams. It is further purified in the second column to obtain the 200 gms of Glabridin with about 95% purity. The purity of the compound is estimated using HPLC, the details of which are as below.

HPLC Method:
The Glabridin content in the powdered extracts recovered from the process was analyzed using an HPLC system consisting of a quaternary pump with a vacuum degasser, thermos tatted column compartment, autosampler, and UV detector. A reverse-phase column (Inertsil ODS, C18, 3V, 5 µm, 250 X 4.6 mm; GL Sciences, Japan) was used with a column temperature of 40 °C. The HPLC mobile phase Solution A: Potassium dihydrogen phosphate (1.36 g) was dissolved in 1000 mL of HPLC grade water. The solution was filtered through a 0.45 µm membrane filter and degassed in a sonicator for 3 min; Solution B: Acetonitrile (100%). The mobile phase was run using gradient elution. The detection wavelength for Glabridin was chosen as 220 nm, based on the UV spectra of pure Glabridin solution. The flow rate was kept as 1.0 mL/minute and injection volume was 20 µL. The run time for detection of Glabridin by HPLC was 65 min. The details of the column are presented in Table 14.
Buffer solution:
1.36 gm of Potassium dihydrogen orthophosphate (KH2PO4) was dissolved in 1000mL of water and mix thoroughly.
Mobile Phase A: Buffer solution.
Mobile Phase B: Acetonitrile.
Diluent: Methanol was used as a diluent.

Table 14: Column particulars:
Column Inertsil ODS, 3V, 250 x 4.6 mm and 5µm
Column Temperature 40°C
Sampler temperature 10°C
Wavelength 220 nm
Flow rate 1.0 mL/min
Injection Volume 20 µL
Run time 65 minutes
Retention Time At about 50.0 min
Elution Gradient

The gradient program for separation of Glabridin by HPLC was set as shown in Table 15.

Table 15: Gradient Program:
Time (minutes) % Mobile phase: A % Mobile phase: B
0.01 95 5
25.00 60 40
40.00 58 42
45.00 40 60
50.00 40 60
55.00 20 80
60.00 20 80
61.00 95 5
65.00 95 5

Pure compound quantity obtained: 200 mg
Purity obtained: 95% by HPLC

Sample preparation
50 mg of Glycyrrhiza glabra extract was precisely weighed and transferred to a 50 mL volumetric flask. About 30 mL methanol was added and the contents were sonicated for 20 min. The volume was then made up to 50 mL with methanol, filtered through a 0.45µm filter and the resulting filtrate was used as a test solution. A stock solution of Glabridin standard was prepared at a concentration of 5.0 mg/mL in absolute methanol. The calibration curves were prepared using solutions of different concentration levels from 10 – 550 µg/mL.

The purity of the compound Glabridin achieved through this process was found to be more than 95 %. The experimental data showing 99.45 % purity of Glabridin is as given below. Besides a high yield of the compound Glabridin was obtained of more than 7 % which is unprecedented. The data explaining Glabridin 7.2% content calculation/yield is as illustrated below.
Standard and sample preparation data for HPLC Purification
Standard preparation: 2.5mg/25mL further, 2mL in 20mL (10 ppm)
Injection volume: 20µL (0.2 µg)
Sample preparation: 25mg/mL (1000 ppm)
Injection volume: 20µL

Area normalization values
Standard area: 474235 for 0.2 µg
Sample area: 3433521 for 1.44 µg (in 20µL)
1.44/3433521×474235=0.1989
Purity = 0.1989/0.2×100=99.45%
Yield 1.44/20×100=7.2 %
Therefore, 100 mg contains 7.2 mg Glabridin i.e. (7.2%) considering 100% pure working standard.

Ingredients of the Pharmaceutical composition (oral Dosage Form)
The pharmaceutical composition for arthritis as per a preferred embodiment of this invention comprises of the ingredients as illustrated in table 16 below.

Table 16: Formulation process and ingredients of pharmaceutical composition
Sr. no Ingredients Mg/cap
1 Glycyrrhiza glabra extract or isolated purified Glabridin 200.00
2 Microcrystalline cellulose (Avicel PH 101) 190.00
Wet granulation
3 Purified water q. s
Blending
4 Colloidal silicon dioxide (Aerosil 200) 5.00
Lubrication
5 Magnesium stearate 5.00
Total 400.00
6 Vcap HPMC size “0” capsules clear cap/clear body 1

Glabridin as an injectable/parenteral formulation:

Corticosteroid injections are therapeutic injections used to relieve joint pain and inflammation. Common corticosteroids used include triamcinolone, methylprednisolone, and dexamethasone and other drugs such as Otilimab.

The present invention relates to injectable formulations of Glabridin for similar purposes.

Disease-modifying antirheumatic drugs (DMARDs) like TNF alpha inhibitors 9adalimumab, infliximab, etanercept, golimumab, and certolizumab pegol), IL-6 inhibitors include tocilizumab and sarilumab, IL-17 inhibitors such as secukinumab, ixekizumab, and ustekinumabcannto be given orally and hence injectable formulations are crucial to formulate.

In the light of the above facts, a liquid formulation of Glabridin was prepared. Since Glabridin is a non-polar compound whose solubility in water is very low, to make it a possible parenteral formulation, inventors have used organic solvents such as propylene glycol and also agents such as cyclodextrins (Kleptose).

The cyclodextrins are the complexing agents used to increase the aqueous solubility of active substances that are poorly soluble in water so that their bioavailability and stability is improved. The three different types of cyclodextrins known are a-cyclodextrin, ß-cyclodextrin, and ?-cyclodextrin that are composed of six, seven, and eight glucose units, respectively. ß-cyclodextrin (ß-CD) is the most commonly used type of cyclodextrins due to its lower price, simpler production, and skin-friendliness and kleptose is one of the examples of the ß-CD.

Table 17:
Glabridin concentration Solubilizing agents Solvent used for dilutions HPLC area (250 ppm) HPLC area (500 ppm)
5 mg/mL Methanol (100%) Methanol (absolute) Soluble
15007284 Soluble
26415142
5 mg/mL Propylene glycol (100%) Water Insoluble
30045 Insoluble
144900
5 mg/mL 100mM kleptose in water Water Soluble
15094895 Soluble
30337889
5 mg/mL Propylene glycol (100%) 5mM kleptose in water Soluble
15654124 Soluble
30568934
5 mg/mL 100mM Methyl beta-cyclodextrin in water Water Not done Soluble
28847709
5 mg/mL 100mM Hydroxyethyl beta-cyclodextrin in water Water Not done Soluble
28949380

Thus, an efficacious and effective parenteral formulation was prepared using purified Glabridin (5 mg/mL) using 100mM kleptose in water, which can further be diluted with Propylene glycol (100%) as solubilizing agent and 5mM kleptose in water as solvent depending on the concentration of glabridin as required for the therapeutic effect against arthritis. This concentration may vary depending upon the body weight, age, gender and other physiological patient parameters.
, Claims:1. A process for extraction of active compounds from Glycyrrhiza glabra roots, comprising of:
a. mixing the root of weight 100-500 grams with five volumes of a solvent in a temperature range of 30-70°C;
b. repeating the cycle of mixing the root with solvent two or more times with each cycle being for a time duration of 2 hours to 4 hours to obtain a liquid mixture;
c. filtering the liquid mixture through filter paper to obtain a liquid extract; and
d. concentrating and drying the liquid extract as obtained from step c using an evaporator, keeping the temperature in the range of 35°C - 60°C to obtain an extract of weight in the range of 10 – 20 grams;
wherein the active compounds extracted are effective in improving conditions of Arthritis by Hexokinase inhibition activity.

2. The process as claimed in claim 1, wherein the solvent used for extraction is Acetone.

3. The process as claimed in claim 1, wherein the weight of Glycyrrhiza glabra roots used in step 1 a is 300 grams.

4. The process as claimed in claim 1, wherein the temperature range in step 1 a is 50 °C.

5. The process as claimed in claim 1, wherein the number of cycles of mixing in step 1 b is at least three and the time duration of one cycle is at least three hours.

6. The process as claimed in claim 1, wherein the temperature used for concentrating and drying is 45-50° C.

7. The process as claimed in claim 1, wherein the weight of extract obtained is 15 grams.

8. The process as claimed in claim 1, wherein one of the active compounds extracted is Glabridin.

9. A method of isolating and purifying Glabridin from an extract comprising one or more additional bioingredients or impurities using a pair of Columns for column chromatography, comprising the steps of:
a. adsorbing the 10-20 grams of extract containing Glabridin along with additional bioingredients or impurities, on the stationary phase of the first column comprising of silica of size 60-120#;
b. subjecting the extract to the stationary phase of the second column comprising of silica of size 100-200#;
c. eluting Glabridin with a first mobile phase;
d. eluting Glabridin with a second mobile phase; and
e. purity estimation of the compound Glabridin using HPLC method; and
f. detecting Glabridin by analyzing the chromatogram from the HPLC method;
wherein the mobile phase comprises one or more solvents, such that the active compound flows through the column with a retention time that is different from one or more other ingredients of the mixture.

10. The method as claimed in claim 9, wherein the solvent used in the first mobile phase is Hexane in the concentration range of 70-100 %.

11. The method as claimed in claim 9, wherein the solvent used in the second mobile phase is ethyl acetate in the concentration range of 1-15%.

12. The method as claimed in claim 9, wherein the solvent used in the first mobile phase is Hexane of concentration 100%.

13. The method as claimed in claim 9, wherein the solvent used in the second mobile phase is ethyl acetate of concentration 8%.
14. The method as claimed in claim 9, wherein purity and yield of Glabridin as obtained is 95%.

15. The method as claimed in claim 9, wherein the details of the phases and process parameters of HPLC method for purity estimation of Glabridin are as shown below:
Mobile Phase First Solvent (A): Buffer solution
Second Solvent (B): Acetonitrile.
Diluent Methanol
Column Inertsil ODS, 3V, 250 x 4.6 mm and 5µm.
Temperature of Mixture in the range of 1-20°C
Temperature of column in the range of 30-50 °C
Wavelength in column in the range of 150-350 nm
Flow rate in column in the range of 0.2-5 ml/min
Injection volume 20 ?L
Mobile Phase run time In the range of 50-75 minutes
Retention time At about 40-60 min
Elution Gradient

16. The method as claimed in claim 15, wherein the buffer solution is formed by dissolving 1.36 gm of Potassium dihydrogen orthophosphate (KH2PO4) in 1000mL of water and mixed well for use.
17. A pharmaceutical composition for arthritis, comprising of:
a. Glabridin extract or isolated purified Glabridin;
b. an additive as a microcrystalline cellulose;
c. an additive as an anti-caking agent;
d. an additive for lubrication; and
e. purified water
wherein the composition is formulated as an oral dosage form for reducing inflammation, hyperalgesia and bone resorption.

18. The composition as claimed in claim 17, wherein the Glabridin extract is acetone extracts or isolated purified Glabridin from Glycyrrhiza glabra.
19. The composition as claimed in claim 17, wherein the anti-caking agent is selected from a group comprising colloidal silicon dioxide (Aerosil 200), magnesium silicate, ammonium chloride, dioxins, non-colloidal MCC etc.

20. The composition as claimed in claim 17, wherein the additive for lubrication is selected from a group comprising magnesium stearate stearic acid, potassium stearate, lithium stearate etc.

21. The pharmaceutical composition as claimed in claim 17, consisting of
Ingredient Concentration Range
Glabridin extract or isolated purified Glabridin; 1 to 400 milligrams
microcrystalline cellulose 1 to 300 milligrams
colloidal silicon dioxide.
as an anti-caking agent; 0.1 to 10 milligrams
Magnesium Stearate for lubrication; and 0.1 to 10 milligrams
purified water q.s

22. A process for preparing a pharmaceutical composition for arthritis wherein the steps of said preparation comprise of:
a. obtaining Glabridin as an acetone extract or as isolated purified Glabridin from the plant source;
b. mixing the Glabridin extract or isolated purified Glabridin with microcrystalline cellulose;
c. conducting wet granulation of the mixture in previous step by adding purified water;
d. conducting blending by adding an anti-caking agent to the above mixture;
e. conducting lubrication using an additive for lubrication; and
f. filling the above mixture in a capsule.

23. A pharmaceutical composition comprising of
a. Isolated purified Glabridin in the concentration range of 3mg/ml to 7 mg/ml; more preferably 5mg/ml;
b. 100mM kleptose (KLEPTOSE® HP Parenteral grade Hydroxypropyl ß-cyclodextrin) or other permissible cyclodextrins of parenteral grade as Solubilizing agent; and
c. Solvent including water for dilutions
wherein the composition is formulated as a parenteral/injectable dosage form for synergistically enhancing the repair of RA joints.

24. A process of preparing a pharmaceutical composition as claimed in claim 23 wherein requisite amount of isolated purified Glabridin is dissolved in predetermined amount of solubilizing agent and then diluted with predetermined amount of solvent to formulate a parenteral/injectable dosage form for synergistically enhancing the repair of RA joints.