During the shodhana process of guggulu, the following facts were observed: (i) initially the liquid was light brown in color and subsequently turned into dark brown; (ii) evaporation was started at 70 °C, which was aggravated on stirring; and (iii) the maximum temperature was recorded up to 90‒100 °C. On average, semisolid guggulu was prepared after 3 h (avg. for three batches). The final yield of guggulu (semisolid) obtained was 680 g (avg. for three batches). The final quantity of purified guggulu residue obtained was 320 g (avg. for three batches). During the purification, it was mandatory that all vessels were non-reactive and cleaned properly before use. During boiling, the liquid should be stirred continuously. Care should be taken to avoid direct product contact.

Table 1 shows that foreign matter, loss on drying or moisture content, pH, ethanol-extractive value, total ash, and acid-insoluble ash of guggulu and shodhita guggulu were significantly different (at p < 0.001). This significant variation in the moisture content was due to the interaction of guggulu with water molecules during its shodhana process. This significant variation in pH might be due to the reduction in the acidic metabolites of guggulu during its shodhana process. It is documented that the shodhana process helps to reduce the gastric irritation effects of guggulu [28, 38]. The water-extractive values of guggulu (61.08% w/w) and shodhita guggulu (63.56% w/w) were non-significantly different (at p < 0.001) which means gowmutra shodhana has non-significant effects on the polar molecules of guggulu. The ethanol-extractive value of guggulu and shodhita guggulu were significantly different which means gowmutra shodhana has significant effects on the polar as well as non-polar molecules of guggulu as ethanol is highly miscible in non-polar solvents like hexane. The increase in total ash and acid-insoluble ash after shodhana confirms the facts and trends observed in water and ethanol-extractive values. As non-significant changes in water-extractive value means the polar molecules including metal ions are remains after shodhana, similarly, the ethanol-extractive value after shodhana was decreased almost to 50% which means that the ratio of polar molecules including metal ions has increased and the ratio of non-polar molecules has decreased. The observed values of λmax of guggulu and shodhita guggulu were 324 nm which was non-significant, meaning that the major molecules of guggulu are not depleted after shodhana (Table 1).

In a HPTLC study, mobile phase optimization is the main phase. Better mobile phase means good resolution and better separation of compounds. In the present study, the mobile phase consisting of toluene‒ethyl acetate‒acetone‒formic acid (8.5:0.5:1:1, V/V) at room temperature (25 ± 2 °C) was optimized for the HPTLC analysis. The observed results of HPTLC analysis for guggulu, shodhita guggulu, and marker compounds (guggulsterone E and guggulsterone Z) are given in Table 1. In the HPTLC analysis, at 254 nm, twelve bands were observed including the bands of the two marker compounds guggulsterone E (RF 0.81) and Z (RF 0.85) (Fig. 1). Marker compounds were not detected at 366 nm and after derivatization of TLC plate (Table 1 and Fig. 1). In the HPTLC analysis at 254 nm, a new band at RF 0.31 was observed after the shodhana of guggulu sample. After shodhana, the intensity as well as the resolution of bands also increased which means fatty organic matter with volatile nature was removed. Further, the pH study results revealed that the removal of organic matters having acidic nature occurred during the shodhana process.

Comparative HPTLC analysis of guggulu (G) and shodhita guggulu (SG) at 254 nm (A), 366 nm (B), and 514 nm (C) w.r.t. their marker compounds. Track 9: guggulu (G), track 10: shodhita guggulu (SG), track 11: guggulsterone E (GE), and track 12: guggulsterone Z (GZ). *New peaks after shodhana. #Intensity or resolution increased after shodhana

There are various studies on the quantification guggulsterone E and Z alone. The developed method is better than recently reported methods for the profiling of guggulsterone E and Z alone. Musharraf et al. (2011) have developed simultaneous quantification of guggulsterone E and guggulsterone Z, where the mobile phase hexane‒ethyl acetate‒formic acid (55:40:5, V/V) was used to quantify guggulsterone E and Z [36]. The basic difference between the present study and the study reported by Musharraf et al. (2011) is the resolution and the HPTLC plate used. In the present study, simple TLC plates were used, whereas Musharraf et al. (2011) used glass HPTLC plates which are costly and not every laboratory can afford it. In terms of resolution, Fig. 1 shows the bands of high resolution that is blade-like sharpness, which is very rare and needs more skills to develop such plates. In another study, the mobile phase toluene‒acetone (9.3:0.7, V/V) was used to quantify guggulsterone E and Z in herbal extract where simple TLC plates were used, but resolution was less than in the present study [37].

To verify and validate the HPTLC analysis results, LC‒QTOF‒MS/MS analysis of guggulu and shodhita guggulu was performed. To attain the significant results, the same concentration of guggulu and shodhita guggulu and the same method conditions were used. The LC‒QTOF‒MS/MS analysis of the methanolic extract of guggulu and shodhita guggulu revealed the presence of 45 compounds (in ESI+) including the presence of the marker compounds, i.e., guggulsterone E and Z were confirmed in the HPTLC analysis (Fig. 2). A detailed literature and library search has confirmed the presence of eight major phytochemicals as indicated by green lines in the chromatograms of guggulu and shodhita guggulu in Fig. 2. Figures 2 and 3 show the presence and fragmentation pattern of 5-(13′Z-nonadecenyl)resorcinol (Rt 8.31), 8β-hydroxy-3,20-dioxopregn-4,6-diene (Rt 10.33), guggulsterone E (Rt 12.99), guggulsterone Z (Rt 13.28), guggulsterol I (Rt = 15.19), mangiferolic acid (Rt = 15.27), 20(S),21-epoxy-3-oxocholest-4-ene (Rt = 15.75), and guggulsterol II (Rt 16.23). Figure 2 shows that between Rt from 4 to 21 min, maximum peaks became sharp and intense in shodhita guggulu sample. Various humps and doublets have been eliminated after the shodhana process.

Comparative LC‒MS chromatograms of guggulu and shodhita guggulu ESI (+ ve) mode; 5-(13′Z-nonadecenyl)resorcinol (Rt 8.31); 8β-hydroxy-3,20-dioxopregn-4,6-diene (Rt 10.33); guggulsterone E (Rt 12.99); guggulsterone Z (Rt 13.28); guggulsterol I (Rt = 15.19); mangiferolic acid (Rt = 15.27); 20(S),21-epoxy-3-oxocholest-4-ene (Rt = 15.75); and guggulsterol II (Rt 16.23). *New peaks after shodhana. #Intensity increased after shodhana. $Intensity decreased after shodhana

Major fragmentations pattern of guggulu and shodhita guggulu observed in LC‒MS ESI (+ ve) mode study; 5-(13′Z-nonadecenyl) resorcinol (Rt 8.31); 8β-hydroxy-3,20-dioxopregn-4,6-diene (Rt 10.33); guggulsterone E (Rt 12.99); guggulsterone Z (Rt 13.28); guggulsterol I (Rt = 15.19); mangiferolic acid (Rt = 15.27); 20(S),21-epoxy-3-oxocholest-4-ene (Rt = 15.75); and guggulsterol II (Rt 16.23)

The compound databases, namely PubChem, KEGG Compound and ChemSpider were used to search the metabolites. Moreover, detailed literature search was executed to confirm the phytochemicals of guggulu and shodhita guggulu as shown in Table 2. The patterns of the elution of phytochemicals were confirmed as almost the same conditions were used by Haque et al. (2009) and Ahmed et al. (2016) [29, 32]. In HPLC study, they confirmed the elution pattern as 5-(13′Z-nonadecenyl)resorcinol, 8β-hydroxy-3,20-dioxopregn-4,6-diene, guggulsterone E, guggulsterone Z, guggulsterol I, mangiferolic acid, 20(S),21-epoxy-3-oxocholest-4-ene, and guggulsterol II; 5-(13′Z-nonadecenyl)resorcinol with m/z 408 [M + H]+ having the fragmentations at 366, 321, and 242. Chhonker et al. (2018) confirmed that guggulsterone E and Z can be fragmented into various m/z ratios after multimechanisms [33]. The main fragmentations of guggulsterone E and Z were m/z 295, 285, 277, 255, 230, 201, 162, 109, and 97 [26, 34]. Hydroxylation process can convert guggulsterone E and Z into molecule having molar mass m/z 329 with main fragmentations 293, 269, and 215. Dihydroxylation process can convert guggulsterone E and Z into molecule having molar mass m/z 345 with main fragmentations 327, 309, 267, and 263. Trihydroxylation process can convert guggulsterone E and Z into molecule having molar mass m/z 361 with main fragmentations 325, 311, and 283. Quaternary hydroxylation process can convert guggulsterone E and Z into molecule having molar mass m/z 377 with main fragmentations 359, 317, 198. Acetylation process can convert guggulsterone E and Z into molecule having molar mass m/z 355 with main fragmentations 337, 327 [29,30,31,32,33]; guggulsterol I with m/z 437 [M + Na]+ having the fragmentations at 423, 413, 359, 309, and 227; mangiferolic acid with m/z 455 [M + H]+ having the fragmentations at 437, 423, 413, 359, 309, 227, 187, and 115; 20(S),21-epoxy-3-oxocholest-4-ene with m/z 417 [M + Na]+ having the fragmentations at 399, 359, 309, and 177; guggulsterol II with m/z 417 [M + H]+ having the fragmentations at 399, 359, 309, 227, and 177 [29,30,31,32,33]. Bhatia et al. (2016) [34] and Kumar et al. (2020) [5] revealed guggulu as a potential source for pharmaceuticals, nutraceuticals, and a wide class of phytochemicals, so no surprise for the presence and identification of biological potent compounds in guggulu.

Guggulu has shown a broad range of activities, but the main activities are anti-hyperlipidemic, anti-inflammation, and anti-arthritic activities [5, 16, 17]. A study has revealed that guggulu helps to reduce the COVID-19-based obesity [21]. Guggulu is exported in more than 42 countries including developed countries like United Kingdom and United State America. The demand for guggulu is more than its production [39]. It has been reported that older guggulu is more potent than fresh guggulu [40, 41]. Further, purification process (shodhana) makes it more potent [39,40,41]. There is a lack of scientific reports where detailed studies on purified and non-purified guggulu are missing. In the present study, cow urine-processed guggulu and raw guggulu were studied by using advanced tools of HPTLC and LC‒MS techniques including the basic parameters as mentioned in API. Under HPTLC study, band resolution was increased with purification and new bands were also noticed, e.g., a sharp and new band at RF 0.31 was noticed in the purified guggulu. Similarly, in LC‒MS/MS study, peak resolution was increased and new peak at Rt 5 min was observed. In Fig. 2, the intensity of peaks was increased and broad peaks of raw guggulu turned into sharp peaks after purification process. There are various guggulu formulations and other formulations are mentioned in the Ayurvedic Formulary of India (AFI) where guggulu is the main ingredient [40]. There are various polyherbal drugs already available in the national and international market [5, 16, 17]. Shodhana of guggulu is the mandatory part of each formulation as mentioned in AFI [5, 39]. There are various methods of shodhana prescribed in AFI [40]. Purification may enhance the activity of guggulu by removing its toxicity. There is no report on gowmutra shodhana (cow urine-based purification) yet. This is the first study where purified and non-purified guggulu were compared by using advanced tools, and major phytochemicals like guggulsterone E, guggulsterone Z, and guggulsterols were identified.