Comprehensive chemo-profiling of coumarins enriched extract derived from Aegle marmelos (L.) Correa fruit pulp, as an anti-diabetic and anti-inflammatory agent

Graphical abstract


Introduction
Ayurveda is an ancient Indian medicinal system which has enlisted and described hundreds of diseases and their cure from medicinal plants. (Chauhan et al., 2015;Kumar et al., 2017;Dass, 1939). One of these medicinal plants, Aegle marmelos (L.) Correa (Family: Rutaceae) is known for its various therapeutic activities, as Rig Veda and Charak Samhita described. Aegle marmelos, commonly known as Bael, Bengal quince or Golden apple, is indigenous to the Indian sub-continent and has spiritual and religious significance in Hinduism (Dass, 1939). In Ayurveda, the unripe fruit is known to balance kapha and vata dosh (Bhardwaj and Nandal, 2015;Dass, 1939). In comparison, the young leaves (Bilva patra) are known to balance all three doshas and relieve pain, dyspepsia, gastritis, and abdominal colic pain (Sarkar et al., 2020). Its roots improve digestion, while the stem is efficacious for rheumatoid arthritis and heart disease (Shastri and Chaturvedi, 2004;Dass Ji, 1939). Aegle marmelos fruit powder is known to be one of the necessary items in Ayurvedic formulations. Protective effects against the wound, radiation, microbes, free radical generation, and depression have also been exhibited by Aegle marmelos. These records prove the natural healing power of Aegle marmelos. (Bhardwaj and Nandal, 2015;Sarkar et al., 2020).
Recent research and clinical studies on the crude extracts of the various plant parts showed the anti-diarrhoeal, anti-microbial, antiviral, anticancer, chemopreventive, antipyretic, analgesic, anti-ulcerative, diuretic, antifertility and anti-inflammatory properties (Choudhary et al., 2021). Aegle marmelos fruit has been reported to contain some valuable phytoconstituents such as marmelosin, marmelide, psoralen, alloimperatorin, rutaretin, scopoletin, aegeline, umbelliferone, marmelin, fagarine, anhydromarmelin, limonene, a-phellandrene, betulinic acid, marmesin, luvangentin and auroptene. A study has reported that the fruit pulp of the plant contains a high quantity of tannins and riboflavins also (Bheeman et al., 2014;Rahman and Parvin, 2014;Reddy and Urooj, 2013;Tiwari et al., 2022). Despite the popularity of this plant, a systematic phytocompound(s) enrichment, chemical characterization, and bioactivity assessment have not been reported simultaneously in the scientific literature. The present study aims to enrich the principal active compounds in Aegle marmelos (L.) Correa fruit pulp, targets antiinflammatory and anti-diabetic activity, leading to the development of a Phytopharmaceutical ingredient (PPI) with a defined dosage based on in silico and in vitro assays. The extracts were characterized and quantified by HPTLC, HPLC, DSA-MS, LC-MS, and qNMR. This study developed the process regarding crude extract enrichment, which can further be implemented for the isolation of compound(s) of interest. By using the advanced in silico approach, the target analytes were evaluated for their druglikeness. In vitro, cytotoxic assay and Scanning Electron Microscopy (SEM) analysis confirm the in silico results. The study is holistic and can be implemented further for drug development and design.

Plant material collection and authentication
For the present study, dried fruits of Aegle marmelos was bought from the local market and authenticated from CSIR-NIScPR (authentication no. -NISCAIR/RHMD/Consult/2021/4160-61-1). The fruits was coarsely powdered and stored in an airtight container for further use.

Extraction and coumarins enrichment
The pharmacognostic tests (loss on drying, total ash, acidinsoluble ash, and water & ethanol -soluble extractive values) of the fruit powder of Aegle marmelos were assessed and then about 5.0 g was accurately weighed and extracted with 50 mL methanol by refluxing at 65°C for 2 h, cooled and filtered. The residue was extracted three times with the same procedure to get maximum yield. The filtrate obtained was combined and concentrated to dryness using a rotary vacuum evaporator, dried in a vacuum oven and extract yield was calculated on the dried basis.
The coumarins enrichment was done by following a method described for marmelosin isolation. After some modifications and many trials, the enrichment process for coumarins was designed (Shinde and Laddha, 2012). A portion of the dried extract was kept for screening studies; the remaining was dissolved in 10% w/v NaCl solution in water and filtered. First, the filtrate was partitioned with ethyl acetate repeatedly, and an organic layer was collected. Subsequently, the aqueous layer was acidified with acetic acid to pH-4 and again partitioned with ethyl acetate, followed by aqueous layer basification with ammonia solution to pH-9 and partitioned with ethyl acetate. Finally, all three organic layers were combined and concentrated to obtain coumarins enriched extract. Further purification was done using Vacuum Liquid Chromatography (VLC), as explained in Supplementary Figure 1.

In vitro anti-hyperglycaemic activity
To determine the anti-diabetic potential of the crude and enriched extracts, a-amylase Inhibition Assay was performed. Briefly, the test samples (in series of 100 to 1000 lg/ml) were added to the a-amylase enzyme and incubated for 10 min at 37°C. Afterwards, the starch solution (1% w/v), i.e., the substrate was added to the mixture and incubated for 10 min. Finally, the DNS reagent was added to the solution and incubated for 10 min at 37°C. The reagent colour changes to orange/deep red in the presence of sugar formation, which was measured by UV-Visible spectrophotometer at 540 nm (Ali et al., 2006;Mishra S.,Khatri M., 2019;Tamil et al., 2010).

In vitro anti-inflammatory activity
Test samples were checked for protease inhibitory response by pre-incubating 1 mL Trypsin (10 U/mL) with 1 mL of a series of samples (100 lg/ml to 1000 lg/ml) at 37°C for 15 min. 2 mL of 1% Bovine Serum Albumin (BSA) (0.1 M phosphate buffer) was added and incubated at 37°C for 30 min. The reaction was terminated by adding 2.5 mL of 0.44 M Trichloroacetic acid (TCA). The reaction mixture was centrifuged at 10,000 rpm for 15 min, and the pellet was discarded. The absorbance of the supernatant was measured at 280 nm (Hashmi et al., 2022;Leelaprakash and Mohan Dass, 2011;Sakat et al., 2010).

In silico studies
For the docking study, the proteins were prepared by Autodock tools, protein docking was done using CBdock, and the proteindrug interaction was visualized by Biovia Studio. The in silico anti-diabetic study was assessed on three proteins viz., aamylase (2QV4), b-glucosidase (2ZOX), and pancreatic lipase (2OXE). The targeted molecules viz., marmelosin, marmesin, aegeline, psoralen, scopoletin, and umbelliferone in Aegle marmelos fruit pulp were docked with the proteins as mentioned earlier along with the standard drugs suitable as the enzyme inhibitors (Supplementary Figure 2). For a-amylase, acarbose was selected; for b-glucosidase -miglitol, and pancreatic lipase -sibutramine was selected. To determine the intestinal absorption, druglikeness, volume distribution, metabolism, renal clearance, and potential hepatotoxicity, the ADMET test was performed using PkCSM software. The molecules were also evaluated for the Lipinski rule of 5 violations by the software provided by IIT-Delhi ''SCFBio." OSIRIS tool was used to evaluate the drug score and Molinspiration for bioactivity factor.

In vitro cell cytotoxicity assay
The cytotoxicity profiling of the crude and enriched extract of Aegle marmelos fruit powder was checked on THP-1 (human monocytic cell lines) and A549 (adenocarcinomic human alveolar basal epithelial cells) cell lines. Cell lines were maintained and cultivated in sterile Biosafety lab-2 (BSL-2) in a CO 2 incubator. For THP-1 and A549 cell lines, RPMI1640 and DMEM media with 10% Foetal Bovine Serum (FBS) with 1% Pen-strep solution (Penicillinstreptomycin solution) was used at 37°C and 5% CO 2 . For the MTT assay, cells were transferred in 96 well plates (5000-7000 cells per well) and incubated with the test samples in serial concentration (100 lg/ml to 1000 lg/ml). After 48 h of incubation, cells were washed 2 times with sterile 1XPBS and 100 ll fresh media was added. 10 ll of MTT dye was added to each well and incubated for 4 h (or till colour developed). Live cells change the yellow-coloured MTT dye into the blue to violet colouration. To stop the reaction and dissolution of the coloured product, 100% DMSO was added to each well (Duraipandiyan et al., 2015;Latha et al., 2018).

SEM analysis on Staphylococcus aureus in the presence of crude and coumarins-enriched extracts
As the anti-inflammatory property of the enriched extract is targeted in the present study, the inflammatory diseases and the bacteria causing the inflammatory disease should be focussed. Staphylococcus aureus is a well-known pathogen which causes severe skin inflammation, showing symptoms like boils, blisters and acne-like rashes. Hence, the effect of the enriched extract was also checked on the bacterium, and SEM analysis was done. S. aureus ATCC 6538 culture was revived, and the working culture was prepared by taking one colony from the streaked plate, inoculating in fresh media, and incubating at 37°C for 24 h. Three sterile test tubes were taken, in which 1 mL bacterial culture (10 8 CFU/mL) was transferred along with 5 mL media; the first one was used as Control, while in the second and third tubes, 0.5 mL of crude and enriched extracts were added at their IC 50 concentration, as determined by in vitro anti-inflammatory activity. All three tubes were sealed and incubated for 48 h at 37°C. After the incubation completion, the cultures were vortexed and centrifuged at 3000 rpm for 10 min. The supernatant was discarded, and pellets were washed thrice with 1XPBS. Briefly, 200 ll of 2.5% solution of Glutaraldehyde was added to the tubes and incubated at room temperature for 4 h and refrigerated (2-4°C) for 48 h. After centrifugation, the solution was removed, washed with phosphate buffer thrice, and reconstituted in the same. The vials were sealed and sent to the Sophisticated Analytical Instrumentation Facility, AIIMS, Delhi, for sample viewing (Mishra et al., 2021).
2.9. Compound(s) characterization in crude and enriched extract 2.9.1. High-performance thin layer chromatography (HPTLC) analysis Stock solutions of 0.02 mg/ml for standards viz. marmelosin and marmesin, aegeline, scopoletin, psoralen, and umbelliferone were prepared in methanol. Mobile phase: 12:8 -Hexane: Ethyl acetate; 10 ll of each solution was applied to a 20 cm Â10 cm silica plate with an aluminium coating (Silica gel 60 F 254 ) as bands of 10 mm Â 2 mm. The mobile phase was allowed to rise 8 cm and examined under ultraviolet light at 254 nm and 366 nm. For quantification, 5 bands of the standard solution prepared were applied serially (1 ll, 2 ll. . ... . .0.5 ll) along with one band each of crude extract (5 ll each) and PPI prepared from Aegle marmelos fruit powder on a 10 cm Â 10 cm plate. The standard curve was plotted, and the standard amount was detected and calculated (Avula et al., 2016).

HPLC analysis
For HPLC analysis, stock solutions of 0.02 mg/ml for standards viz. marmelosin and marmesin, aegeline, scopoletin, psoralen, and umbelliferone and 1 mg/ml solution of crude and enriched extracts were prepared in methanol. 10 ll each of the standard and the test solutions were injected into the HPLC system, Agilent 1260 infinity, connected with a C 18 column (Orbit) of 150 mm Â 4.6 mm and 5 lm pore size. Mobile phase: A. water with 0.1 per cent v/v of glacial acetic acid; B. methanol with 0.1 per cent v/v of glacial acetic acid; column temperature: 35°C; at 254 nm with gradient program. (Supplementary Table 1).

Direct sample analysis by mass spectrometry (DSA-MS)
The crude and enriched extract of Aegle marmelos fruit was subjected to Direct Sample Analyzer MALDI-ToF, Perkin Elmer by putting one drop of sample solution onto the mesh and analyzed in 0-900 m/z range (Avula et al., 2016).

LC-MS evaluation of crude and enriched extract of Aegle marmelos fruit pulp
For LC-MS analysis, the crude and enriched extract (1 mg/ml) was subjected to Agilent LC-MS-ToF; with C 18 column (Orbit) of 150 mm x 4.6 mm and 5 lm pore size. Mobile phase: A. water with 0.1 per cent v/v of formic acid; B. methanol with 0.1 per cent v/v of formic acid; column temperature: 35°C; injection volume -5 ll with the gradient flow described in HPLC method. The obtained peaks were analyzed according to the online MS library on Pub-Chem CID.

1 H NMR analysis of crude and enriched extract of Aegle marmelos fruit pulp
The crude and enriched extract of Aegle marmelos fruit were subjected to 1 HNMR analysis. 40 mg of the samples were dissolved in 500 ll Deuterated Dimethyl sulfoxide (DMSO) and subjected for 1 HNMR analysis on Jeol JNM-ECZ 400S, 400 MHz; Pulse program -zg30; Solvent -DMSO-D6; No. of scans -40; delayed scan -2; Relaxation time -4 s; Receiver gain -101; Acquisition time -4 s; Pulse width -16.7 W. Benzoic acid was taken as an internal calibrant. The peaks of the targeted compounds were matched with the individual spectrum of the compounds and integrated accordingly to determine the per cent content of respective compound(s) in the crude and coumarins enriched extracts.

Statistical analysis
All analysis was done in a triplicate (n=3), represented as Mean ± SD. The HPLC software used was EZchrome Elite, WinCats for HPTLC. GraphPad Prism 5 was used for plotting graphs and calculating two-way ANOVA and significance was measured in terms of p-value. For the docking study, Chemdraw, PDB data bank, Pub-Chem, CB-dock, PkCSM, SCF-Bio, Biovia Studio, mgltools and other online support and freeware were used. NMR data was interpreted by using Mestre Nova software.

Pharmacognostic studies
Aegle marmelos fruit pulp powder had 5.78% moisture content, 59.23% water soluble extractive and 35.86% ethanol soluble extractive. It was observed that the fruit contains 2.27% and 0.87% of total ash and acid-insoluble ash, respectively, complying with USP-22. Approximately 22.87% yield of the crude extract while 4.87% yield of coumarins enriched extract was noted. The crude extract showed the presence of alkaloids, phenols, flavonoids, tannins, terpenoids, glycosides, and phytosterols. While only alkaloids, phenols and tannins were detected in enriched extract. (Supplementary Table 2).

In vitro anti-hyperglycaemic activity
The crude and enriched extract of Aegle marmelos fruit showed profound anti-hyperglycaemic activity. The crude extract showed lower activity (IC 50 68.35±2.48 lg) compared to the enriched (IC 50 23.85±0.78 lg), indicating enrichment of anti-diabetic agents.

In vitro anti-proteinase activity
Both crude and enriched extracts exhibited anti-proteinase activity, 79.85% and 96.25% proteinase enzyme inhibition at 100 lg/ml. The anti-proteinase activity comparison was made with ibuprofen, showing 97.45% enzyme inhibition at 100 lg/ml. The IC 50 value for ibuprofen, the crude and enriched extract was 35.7 7±0.38 lg, 60.79±1.39 lg and 38.21±0.44 lg, respectively.
3.4.1.2. b-glucosidase. All the target analytes and the standard drug miglitol were targeted on the b-glucosidase enzyme 2ZOX protein.

ADMET study
All targeted molecules followed Lipinski's rule of 5, making them suitable drug candidates. Marmelosin showed the highest intestinal absorption with no hepatotoxicity and a high clearance rate. However, the compound was observed to have low OSIRIS drug-likeness and VDss scores, indicating a lower drug volume distribution. Although it was found to be the substrate of CYP3a4, it is non-inhibitory, showing its degradation by mitochondrial enzymes. Similarly, the molecules were found to be easily permeable to the skin and also through colon cells. All molecules except aegeline were found to be non-mutagenic, whereas umbelliferone was found to be hepatotoxic. The maximum recommended daily dose of marmelosin was found to be 21.5 mg/kg bw/day. While scopoletin and umbelliferone were predicted to be 17.4 mg/kg bw/day and 13.6 mg/kg bw/day, respectively. In the case of aegeline, psoralen, and marmesin, the dosage was found to be 2.68 mg/kg bw/day, 1.6 mg/kg bw/day, and 1.61 mg/kg bw/day, respectively (Figure 7; Supplementary Table 5).

In vitro cell cytotoxicity assay
From the MTT assay conducted on the two cell lines viz., THP-1 and A549, cytotoxicity of the crude and enriched extracts was significantly lower than the standard NSAIDs drug -Paracetamol. The  crude extract was more cytotoxic than the coumarins-enriched extract, confirming the ADMET predictions. It was found that the IC 50 of paracetamol on both cell lines were the lowest (129.50 ± 0.97 lg and 28.50 ± 2.23 lg for THP-1 and A549 cell lines, respectively), which makes it more cytotoxic than the crude (306.18 ± 2.83 lg and 448.39 ± 1.76 lg for THP-1 and A549 cell lines respec-   tively) and enriched extracts (336.18 ± 1.38 lg and 618.26 ± 0.79 lg for THP-1 and A549 cell lines respectively). The difference between the IC 50 value of the standard drug and the extracts is very significant according to the two-way ANOVA test conducted between the samples and the standard. Moreover, paracetamol was more toxic for A549 cell lines with IC 50 concentration lower than on THP-1 cell lines. Coumarins enriched extract exhibited lower cytotoxic on A549 cell lines as its IC50 value is higher on A549 than THP-1. The crude extract of Aegle marmelos fruit was found to have an almost equivalent effect on both cell lines, as observed from its IC 50 value. (Figure 8).

SEM analysis on S. aureus in the presence of crude and enriched extracts
From the SEM analysis, crude and coumarins-enriched extracts of Aegle marmelos fruit showed an anti-bacterial effect on S. aureus. In the control sample, the bacteria were growing in clusters; in the presence of test samples, growth was inhibited and isolated. In the presence of crude extracts, some cells were intact, but most had disfigured cell walls, and holes with cytoplasmic leakage, confirming the cell death. In the presence of enriched extract, all cells were found dead, necrotic, with heavy cell wall damage, as observed at 10,000 magnifications, confirming the antibiotic nature of the enriched extract. This also shows that the enriched extract may have a bactericidal effect as it is causing permanent damage to the cells, hence having a high probability of being used as prescription medicine in upcoming years (Figure 9).

Discussion
Since prehistoric times, humans have been utilizing and repurposing medicinal plants for disease treatment (Wachtel-Galor and Benzie, 2011). With time and the uprisal of modern medicines armed with science and technology, several synthetic drugs have overshadowed traditional knowledge (Singh, 2010). Although causing specific pharmacological effects, synthetic drugs also result in some unavoidable adverse effects. Hence, researchers are moving towards herbal medicines and formulations in the current scenario (Joshi et al., 2011;Sen and Chakraborty, 2017). The sudden increase in the demand for herbal medicines raises issues regarding their safety and efficacy, impeding their acceptability by Indian Medical Association (IMA) and at the global level as mainstream medicines (Ekor, 2013;Katiyar, 2019). The global acceptability for herbal medicines can only be reached if these are developed scientifically. To fulfil this purpose, the Government of India has designed a concept called ''Phytopharmaceutical drugs." Phytopharmaceutical drugs include the purified fraction (s) of the medicinal plant or its part with scientifically proven bioactivity (Bhatt, 2016;Katiyar, 2019). The purified fraction of the plant or its part is considered as a phytopharmaceutical drug if quantification and characterization of a minimum of four marker compounds are done. The chosen marker compounds should contain at least one bioactive marker with defined pharmacological activity (Katiyar, 2019). Our research study is based on the above-mentioned concept, providing a scaffold for phytopharmaceutical drug development.
The present study has developed the coumarins enrichment process in Aegle marmelos, which has successfully enriched bioactive compounds such as marmelosin, marmesin, aegeline, psoralen, scopoletin, and umbelliferone. The method developed is simple, sophisticated and economical, which can easily be implemented at the industrial level. Several research studies have been carried out regarding the isolation of coumarins, which are known bioactive compounds in Aegle marmelos (Chakthong et al., 2012;Manandhar et al., 2018;Shinde and Laddha, 2012;Tiwari et al., 2020). However, the methodologies described earlier for the coumarins compound(s) isolation are complicated and cannot be applied at a large scale (Chakthong et al., 2012). We have depicted and developed a simple liquid-liquid partition technique combined with VLC, easing the separation process with a good percentage yield. The total coumarins were isolated in three easy steps. First, crude extract preparation from Aegle marmelos fruit pulp; second, liquid-liquid partitioning of the aqueous solution of the concentrated methanolic extract with ethyl acetate; and third, separation of the coumarins-enriched extract by VLC.
Previously, several in vitro and in vivo studies have been conducted recurringly on different Aegle marmelos fruit extracts. Out of these, many emphasize the anti-diabetic and antiinflammatory activities of the plant fruit (Arul et al., 2005a(Arul et al., , 2005bKesari et al., 2006;Rajeshkannan et al., 2014;Sabu and Kuttan, 2004;Sudharameshwari and Radhika, 2007;Vyas A et al., 2011). The present study has demonstrated the anti-diabetic and anti-inflammatory activities of the coumarins-enriched extract isolated from the Aegle marmelos fruit, accentuating their potential Fig. 7. ADMET study of all coumarins targeted for enrichment in A. marmelos fruit extract. A) represents drug-likeness score, B) represents intestinal absorption of molecules, C) represents total renal clearance, D) represents molecules absorption in colon, E) represents volume distribution of the target analyte, and F) represents maximum daily dose recommendation for human beings per kg body weight.
R. Tiwari, S. Mishra, G. Danaboina et al. Saudi Pharmaceutical Journal 31 (2023) 101708 use. It was found that the coumarins-enriched extract isolated from the Aegle marmelos fruit greatly inhibits a-amylase and proteinase enzymes, indicating a potent anti-diabetic and antiinflammatory agent. The bioactivity of the enriched extract was greatly enhanced when compared with the crude extract emphasizing the isolation of potent bioactive components. The study also proves its activity is significantly better than the renowned antiinflammatory and hypoglycemic drugs -ibuprofen and metformin. The difference in the IC 50 value of the enriched extract and the standard(s) was found to be significant (p-value 0.01).
Our research further elaborates in silico molecular docking of three main proteins: -a-amylase, b-glucosidase, and pancreatic lipase, which plays a key role in upregulating diabetic symptoms. A total of six coumarins, along with the standard drugs specific for the inhibition of the enzymes, were docked. The results showed that the coumarins of Aegle marmelos have a better affinity with the enzymes than that of the standard anti-diabetic drug. It was observed that the pocket volume in the case of a-amylase enzyme, for the coumarins were larger compared to acarbose indicating a lock and key type of interaction. While for b-glucosidase and pancreatic lipase enzymes, the pocket size is smaller than miglitol and sibutramine respectively, from which we can surmise that they may have better affinity and the interaction might be induced-fit type (Chen et al., 2014;Gao and Skolnick, 2012;Meng et al., 2011). The NSAIDs used for treating inflammation are Cyclooxygenase (COX) inhibitors (Lehmann and Beglinger, 2005;Zarghi and Arfaei, 2011). Some of these specifically inhibit the COX-2 enzyme. COX enzymes are known to produce proinflammatory products, which leads to inflammation. Although the enzyme is known to act upon the chronic inflammation caused due to the diseases, it is also known to cause heart stroke by creating prostanoids imbalance in the renal blood (Sharma and Jawad, 2005;Wright, 2002). Therefore, we chose a new target proinflammatory enzyme -LOX-2, which limits the metabolism of arachidonic acid into leukotriene, mediating inflammation. The enzyme is also known to be actively involved in chronic respiratory disease, atherosclerosis and cancer (Dobrian et al., 2011;Fiorucci et al., 2001;Hu and Ma, 2018;Natarajan and Nadler, 2004). Hence, the coumarins of Aegle marmelos were docked along with the standard NSAID ibuprofen on LOX-2 enzyme. Our in silico docking results showed a very high affinity for the enzyme, with active binding size, indicating an induced-fit interaction. The binding energy of standard NSAID was found higher binding energy, showing lower affinity, hence poor inhibition. Overall, all the coumarins have outperformed the standard anti-diabetic and anti-inflammatory drugs -indicating themselves as promising lead compounds. Their cumulative effect will be synergistic and therefore are precursors for phytopharmaceutical ingredients development.
Further, we have analyzed the in silico ADMET effect as well as the in vitro cytotoxicity evaluation to elucidate adverse reactions (if any) caused by the compounds targeted for enrichment from Aegle marmelos fruit. It was observed that the six coumarins present in the enriched extract, showed overall good absorption, but low volume distribution. The molecules have no cytotoxic effects with excellent skin permeability, which indicates that the gel formulation containing the compounds should work effectively. A slight precaution is needed concerning the content of aegeline and umbelliferone, which showed mutagenic and hepatotoxic effects at higher dosage. This is to be noted that in the enrichment process developed in our study, the content of aegeline and umbelliferone was detected to be 0.72 % w/w and 1.71 % w/w only. Hence, the method developed for coumarins enrichment is significant for industrial purposes. The in silico ADMET results predictions have been confirmed by in vitro cytotoxic assay carried out on the THP-1 and A549 cell lines. The enriched extract of Aegle marmelos was found non-toxic as the IC 50 on these cell lines was found to be more than 500 lg, while for the NSAIDs, it was less than 100 lg, which is significantly higher. This indicates the nontoxic nature of the coumarins-enriched extract.
Diabetes is known to cause chronic inflammation as well as weaken the immune system of the patient, facilitating several health complications (Guthrie and Guthrie, 2004;Kaveeshwar and Cornwall, 2014;Olokoba et al., 2012;Varsha et al., 2018;Wilson, 2007). The increased risk in infection during diabetes is commonly observed in the individual as skin and soft tissue infection (SSTI) (Suaya et al., 2013;Zhou and Lansang, 2021). The causative agent for the infection is Staphylococcus aureus (Thurlow et al., 2020). Therefore, the enriched extract was studied with S. aureus to observe its effect on the pathogen. Through SEM analysis, it was observed that the coumarins-enriched extract causes bacterial inhibition by disrupting their cell wall, causing cytoplasm leakage. Most cells were found shrunk due to the loss of cytoplasm or necrosis, indicating cell death. This also proves the enriched extract isolated from Aegle marmelos will be effective against diabetic foot ulcers (Suaya et al., 2013).
The coumarins-enriched extract isolated from the Aegle marmelos was found to contain approximately 50% of the total coumarins content as a sum total of marmelosin, marmesin, psoralen and scopoletin. Marmelosin was found to be the predominant compound in the fraction with more than 30% w/w content. Following this, around 9% w/w marmesin, 4% w/w psoralen, and 2% scopo-letin was also quantified. A trace amount of aegeline and umbelliferone were also found, whose content was less than 0.70% and 2.0% w/w of the total extract. The content of the coumarins was quantified by three different high-throughput methods, viz., HPTLC, HPLC, and 1 H NMR.
The focus of the present study is the development of the Indian Pharmacopoeia Phytopharmaceutical Ingredient-Reference Standard (IP-PPI-RS) of Aegle marmelos by characterizing and quantifying the six major and valuable bioactive markers with known therapeutic potential. A novel method with simultaneous analysis of all the coumarins was developed which will be highly valuable for researchers and pharmaceutical industries. Indian Pharmacopoeia Commission (IPC) has taken the lead in developing the phytopharmaceutical ingredients reference standards, which may be competing with other pharmaceutical drugs in upcoming years. Indian Pharmacopoeia is giving long-standing recognition to plantbased drugs by developing and validating the PPI monographs and confirming their suitability and permeability for every phytopharmaceutical industry.

Conclusion
The present study entails a simple, sophisticated and inexpensive method of six major bioactive compound(s) enrichment from Aegle marmelos fruit. A targeted approach was taken for bioactive markers selection and their quantification in the enriched extract. The study is supported by implying multiple techniques for the detection and validation of the compound(s) content, making it more reliable. Simultaneous quantification of the targeted coumarins by HPTLC and by other techniques, viz., -HPLC and 1 H NMR, was found to be precise, adding a new dimension in compound (s) characterization in plant extracts. On the basis of in silico and in vitro assays, enriched extract of Aegle marmelos fruit was found to be a potent anti-diabetic and anti-inflammatory agent, withmarmelosin and marmesin showing the best activities against the enzymes targeted for the above-mentioned assays. This study is a major milestone which acts as a scaffold for the phytopharmaceutical drug. The depicted coumarins-enriched extract is an intermediate product before the phytopharmaceutical drug(s) formulation.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.