BIBECHANA Vol. 20, No. 2, August 2023, 175–182 ISSN 2091-0762 (Print), 2382-5340 (Online) Journal homepage: http://nepjol.info/index.php/BIBECHANA Publisher:Dept. of Phys., Mahendra Morang A. M. Campus (Tribhuvan University)Biratnagar In-vitro Dissolution Study of Gallstone with Medicinal Plant Extracts Bijaya B.K.1, Achyut Adhikari1,∗ Gobinda Gyawali2 1Central Dept. of Chemistry, Tribhuvan University, Kirtipur 44618, Kathmandu 2Department of Fusion Science and Technology, Sun Moon University Tangjeong Myeon, Asan Si, Chungnam 31460, Republic of Korea ∗Corresponding author. Email: achyutraj05@gmail.com Abstract Background: Gallstone disease poses a substantial economic burden on healthcare systems globally, necessitating safer alternatives to current treatments like dissolution therapy and cholecystectomy. Natural compounds from plants offer a potential solution, but research on their cholelitholytic activity is limited. In vitro dissolution studies are crucial for identifying effective plant-based therapies. Ob- jective: This study aims to investigate the in vitro cholelitholytic activity of six plants and Ayurvedic medicines, selected based on ethnopharmacological knowledge and folk medicinal practices. Methods: Gallstone samples were categorized as combined cholesterol gallstones (CCGS) or black pigment gall- stones based on external morphology and cross-sectional analysis. In vitro dissolution studies were conducted using extracts from Bergenia ciliata, Berberis asiatica, Cuscuta europaea, Kalanchoe pin- nata, Teraxacum officinale, Macrotyloma uniflorum, and Ayurvedic medicines (Cystone®, Gokshu- radi, and Calcury). The samples were immersed in the extracts and controls separately and incubated in a shaking water bath. The gallstone dissolution capacity was assessed by recording the dry weight of the samples at multiple time points. Results: T. officinale was highly effective in dissolving black pigment gallstones, while B. asiatica exhibited superior efficacy for CCGS. M. uniflorum and C. eu- ropaea also demonstrated significant dissolution activity against black pigment gallstones. However, K. pinnata was less effective for both gallstone types. B. ciliata and C. europaea exhibited equal effectiveness against both types. Ayurvedic medicine extracts were less effective compared to plant extracts. Conclusion: This in vitro study showed the plants can dissolve GS effectively. However, the effectiveness of the plant to dissolve GS depends on the type of the stone. The findings from this study serve as a basis for further in vivo research. Keywords Gallstone, in vitro dissolution, plant extracts, combined cholesterol gallstones, black pigment gall- stones. Article information Manuscript received: June 4 2023; Accepted: June 17, 2023 DOI https://doi.org/10.3126/bibechana.v20i2.55865 This work is licensed under the Creative Commons CC BY-NC License. https://creativecommons. org/licenses/by-nc/4.0/ 1 Introduction Gallstones (GS), which are solid deposits formed in- side the gallbladder, are mainly composed of choles- terol and bilirubin [1]. The formation of GS in- volves supersaturation, nucleation, crystallization, and aggregation of the insoluble components of the bile like cholesterol and bilirubin [2]. When the 175 http://nepjol.info/index.php/BIBECHANA achyutraj05@gmail.com https://doi.org/10.3126/bibechana.v20i2.55865 https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ Bijaya B.K. et al./ BIBECHANA 20 (2023) 175-182 176 concentration of these insoluble components ex- ceeds their solubility or there is a decrease in bile acid concentration or the presence of foreign sub- stances, gallstones form [3]. Gallstone disease is a prevalent health problem that affects 10-20% of the global population [4] and is a significant contributor to morbidity and mortality [5], with an estimated global cost of $6.5 billion annually [6]. Its preva- lence in Nepal is 4.87% with females being more af- fected than males [7]. Therefore, gallstone disease imposes a significant economic burden on health- care systems worldwide [8]. The treatment of gallstone disease can be done through surgery or non-surgical methods. Non- surgical methods include taking bile acids like chen- odeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA) orally (oral dissolution therapy) [9] or installing litholytic solvent like methyl tert-butyl ether (MTBE) [10], 2-methoxy-6-methylpyridine (MMP) [11], or ethylenediaminetetraacetic acid (EDTA) [12], directly into the gallbladder through a percutaneous transhepatic catheter (contact dis- solution therapy) [13]. However, these methods have limitations due to side effects, toxicity, low ef- ficiency [14], incomplete dissolution [15], and gall- stone reoccurrence is common [16]. Laparoscopic cholecystectomy, the surgical removal of the gall- bladder, is considered the best option [17] but has postoperative complications such as bile leakage, bile duct injury [18], persistent pain [19], and fat intolerance [20]. Due to the limitations and po- tential risks of current treatment options, natural compounds from plants could be a safer alternative. It is believed that herbal medicines have lesser or no side effects. While extensive studies have been conducted on the cholelitholytic activity of organic solvents, research on plants is limited, and in vitro dissolution studies are necessary to identify poten- tial plant-based therapies. Therefore, in this study, we investigated the in vitro cholelitholytic activity of six plants (selected based on ethnopharmacolog- ical knowledge and folk medicinal practices) and Ayurvedic medicines available in the market. The results of this in vitro study will provide a basis for further research in vivo. 2 Materials and Methods 2.1 Collection and Pre-treatment of Gall- stones Dr. Barun Kumar Shah, Norvic International Hos- pital, Thapathali, Kathmandu, Nepal has provided the gallstone samples. Cholecystectomy was per- formed to extract gallstones from patients. Alto- gether, 12 CCGS (from two patients) and 15 black PGS (from one patient) were collected. The col- lected gallstones were thrown away materials with no human tissues or genetic material. The gall- stone samples were washed with deionized water and dried at 60 ℃ in an incubator until the con- stant weight [21]. 2.2 Macroscopic Classification of Gall- stones Based on the external morphology and internal cross-sectional analysis, we categorized the gall- stones into two groups: combined cholesterol gall- stone (CCGS) and black pigment gallstone (black PGS). Photographs of the gallstones were taken, their morphological features like shape, size, color, and internal cross-section were studied. The outer dimensions of the stone were measured with a digi- tal vernier caliper. One gallstone from each patient was cut into equal halves using Jeweler’s saw and the internal cross-section was studied. The remain- ing gallstones were stored on a desiccator filled with calcium chloride in a dark cabinet, and later on, they were used for in vitro dissolution with differ- ent extracts of plant and herbal medicines. 2.3 Collection of Plant and Ayurvedic Medicines The rhizomes of Bergenia ciliata were collected from Central Nepal, Parbat District, Panchase. The root of Berberis asiatica and tendrils of Cus- cuta europaea were collected from Central Nepal, Parbat District, Modi rural municipality-7, Ranpu. The leaves of Kalanchoe pinnata were collected from Central Nepal, Kaski District, Pokhara-15, Nayagaun. Leaves of Teraxacum officinale were collected from Central Nepal, Kathmandu Dis- trict, Kirtipur. The legumes of Macrotyloma uni- florum were purchased from the local market of Kirtipur, Nayabazar. Herbarium samples of each plant were prepared and the plants were identi- fied from National Herbarium and Plant Labora- tories, Godawari, Lalitpur. Ayurvedic medicines (Cystone®, Gokshuradi, and Calcury) were pur- chased from Arogyadham Homoeopathic Hospital, Pokhara with the suggestion of homeopathic physi- cian Dr. Bishnu Prasad Chapagain. The plant ma- terials were washed with tap water, cut down into small pieces, and shed dried for two weeks. The shed dried plant samples were crushed into powder with the help of a grinding mill at the Central De- partment of Chemistry. The powder samples were put into an airtight glass jar and stored at ambient temperature. 2.4 In vitro Dissolution Study of Gall- stones The dried and powdered plant samples were ex- tracted with ethanol using Soxhlet apparatus. To Bijaya B.K. et al./ BIBECHANA 20 (2023) 175-182 177 obtain crude extracts, 50 g sample powder was re- fluxed with 500 mL of ethanol at 40 °C until a clear solution was obtained in the thimble. The dilute extract was evaporated under reduced pressure us- ing a rotatory evaporator at 40 °C. The semisolid crude extract obtained from rotavapor was dried at ambient temperature for several days to get the dry crude extract. The dried crude extracts were col- lected into 15 mL flat-bottom borosil culture tubes, labeled properly, and stored at 4 °C in a refriger- ator. 50 mg/mL extract solution was prepared by dissolving 5 g of the solid crude extract in 100 mL of distilled water. The solution was slightly warmed and sonicated for half an hour, let to settle down to get a clear stock solution. The clear solution was collected into a 100 mL volumetric flask, the flask was stoppered, labeled, and stored in a refrigerator at 4 °C. Two tablets of each ayurvedic medicine were powdered in a mortar and pestle and the powder was taken into the centrifuge tube with a screw cap. Distilled water (10 mL) was added and the content was kept in a water bath for a night at 37 °C, centrifuged at 25 °C in a high-speed centrifuge at 8000 rpm to get clear supernatant. The clear su- pernatant was collected into a 100 mL volumetric flask, the flask was labeled and stored at 4 °C in a refrigerator [22]. 2.5 Dissolution of Gallstones with Plant and Medicine Extracts For the in vitro dissolution study, the protocol used by Igimi et al. [23] was followed with slight modifi- cation. The gallstone samples that were oven-dried (at 60 ℃) and stored in a desiccator in a dark cab- inet at least for 15 days were taken for dissolution. The gallstone samples were taken, weighed, and im- mersed in vitro into 10 mL of the extract, positive control, and negative control separately. Borosil culture tubes (15 mL volume) with round bottom and screw cap were used for the purpose. The tubes with content were put in a test tube rack and kept in a shaking water bath at 37 ℃ with gentle and constant shaking (60 rpm). The plant extracts were changed every day to minimize any effect due to sat- uration of the solution by dissolved components of the stone [23]. The stones were taken out of the ex- tract by filtration through filter paper (Whatman No1), washed with distilled water, transferred into a pre-weighed crucible, dried at 60 °C in an incu- bator for two days to get constant weight, and the weight reduced was noted [11,24]. To determine the gallstone dissolution capacity of each extract and solvent, we recorded the dry weight of the stone samples at three different time points (4, 94, and 190 h) after they were directly immersed in the so- lution. Photographs of the stones were also taken after each time interval. The stone samples with comparable weight and size were taken for dissolution study. The average weight of different CCGS samples was 14.48±0.21 mg and that of black PGS samples was 21.46±0.22 mg. In the present research, 2% EDTA solution maintained at pH 9.5 adjusted with HCl and 95% ethanol was used as a positive control whereas dis- tilled water was used as a negative control. It was found that the higher the pH of the EDTA solution higher is its litholytic activity [25]. The pH of 8.5 is within the harmless range to use to the human body [25], but we maintained the pH 9.5 because studies have found that this is the most effective pH at which EDTA shows the maximum dissolution of GS [26]. First of all, the weight dissolved by negative control at every time point was subtracted from the weight reduction value recorded for each of the preparations at respective time points to calculate the actual weight dissolved by the preparations. Then the percentage dissolution was calculated by using the following formula [11, 24]: %Dissolution(w/w) = Actual wt. dissolved Initial wt. of the stone ×100 (1) 3 Results 3.1 General Observation and Macroscopic Classification of Gallstones The photographs showing the external morphol- ogy and internal cross-section of the gallstone sam- ples are given in Figure 1. The gallstone sam- ple, which is multifaceted, whitish-brown with a hard and smooth outer surface [27, 28], with a dis- tinct inner pigmented yellow core and a white ex- ternal shell (Figure 1a, b) [28, 29] was classified as a combination cholesterol gallstone (CCGS). The gallstone sample, which was irregular with a rough and thin yellowish surface [27, 28], amorphous in cross-section [28, 29] with a black inner part and outer thin yellowish layer (Figure 1c, d) was clas- sified as black pigment gallstone (black PGS) [28]. Furthermore, we conducted a separate study to sup- port this macroscopic classification via UV-vis and SEM-EDS analysis [28]. 3.2 In vitro dissolution study of gallstones The final data obtained for cumulative dissolution of gallstone samples in different extract and solvent preparations is presented in Table 1. The obser- vation tables for recorded weight (initial, after 4 h, 94 h, and 190 h), dissolution and cumulative dissolution are included in Supplementary material Bijaya B.K. et al./ BIBECHANA 20 (2023) 175-182 178 Figure 1: Photographs of the gallstone samples; (a) CCGS, (b) Internal cross-section of CCGS, (c) Black PGS, and (d) Internal cross-section of black PGS (Annexure 1). Photographs of gallstones at differ- ent time are also given in Supplementary material (Annexure 2). After 190 hours, 14.3 mg of the CCGS and 8.6 mg of the black PGS was dissolved in EtOH, whereas in EDTA, 11.5 mg of the black PGS and 5.1 mg of CCGS was dissolved. There- fore, it can be inferred that the CCGS was more soluble in EtOH and black PGS was more soluble in EDTA (Table 1). Figure 2 illustrates the com- parative study of the final dissolution of gallstone samples in different extract and solvent prepara- tions. It shows that E1 (M. uniflorum), E4 (C. europaea), E5 (T. officinale), and EDTA dissolved black PGS more effectively than CCGS, while the M2 (Calcury) showed almost similar dissolution for both the stones. In contrast, E2 (B. asiatica), E3 (B. ciliata), M1 (Cystone), M3 (Gokshuradi), and EtOH showed better dissolution for CCGS than for black PGS, while in E6 (K. pinnata), dissolution of both the gallstones was comparable. Table 1: Comparative study of the final dissolution of CCGS and black PGS in different extract and solvent preparations. Extract or Solvent Weight dissolved after 190 hours (mg) CCGS Black PGS E1 (M. uniflorum) 5.6 9.3 E2 (B. asiatica) 10.8 5.4 E3 (B. ciliata) 4.7 3.7 E4 (C. europaea) 7.3 8.5 E5 (T. officinale) 3.0 9.6 E6 (K. pinnata) 2.2 2.1 M1 (Cystone®) 3.1 1.8 M2 (Calcury) 3.9 4.0 M3 (Gokshuradi) 5.8 3.5 EDTA (+ve control) 5.1 11.5 EtOH (+ve control) 14.3 8.6 Note: EDTA and EtOH are used as positive controls and distilled water as a negative control. The data for each experiment is expressed after subtracting the data obtained for the negative control. 4 Discussion Close interpretation of the graph in Figure 2 shows that, in E1 (M. uniflorum), E4 (C. europaea) and E5 (T. officinale), black PGS was more soluble than CCGS. Among different extracts preparations (both plant and medicine), E5 (T. officinale) was the most effective for dissolving black PGS (9.6 mg). In comparison with the most effective pos- itive control EDTA (11.5 mg), the efficacy of E5 (T. officinale) to dissolve black PGS can be con- sidered as comparatively excellent. E5 (T. offici- nale) was more effective than another positive con- trol EtOH (8.6 mg). The efficacy of E1 (M. uni- florum) to dissolve black PGS (9.3 mg) was almost equivalent to that of the most effective plant E5 (T. officinale). This shows that E5 (T. officinale) and E1 (M. uniflorum) can effectively dissolve pig- Bijaya B.K. et al./ BIBECHANA 20 (2023) 175-182 179 Figure 2: The comparative study of the final dissolution of CCGS and black PGS in different extract and solvent preparations ment containing stones. Moreover, this fact was further supported by the result obtained for choles- terol predominant CCGS which shows that E5 (T. officinale) and E1 (M. uniflorum) dissolved signifi- cantly lesser amount of CCGS than black PGS (3.0 mg in E5 and 5.6 mg in E1). M2 showed almost similar efficacy to dissolve both the CCGS (3.9 mg) and GSB (4.0 mg) whereas M1 (Cystone®) and M3 (Gokshuradi) showed better dissolution for CCGS than black PGS. Similarly, E2 (B. asiatica) and E3 (B. ciliata) dissolved CCGS more effectively than black PGS. The efficacy of E6 (K. pinnata) was sim- ilar for both the stones (2.2 mg CCGS, 2.1 mg black PGS). A close inspection revealed that E6 (K. pin- nata) was the least effective plant to dissolve both the CCGS and black PGS. Among different plant extracts, E2 (B. asiatica) was the most effective (10.8 mg) to dissolve CCGS. In comparison with the most effective positive control EtOH (14.3 mg), the efficacy of E2 (B. asiatica) to dissolve CCGS can also be considered significant. E2 (B. asiat- ica) was found to be more effective than another positive control EDTA (5.1 mg). Moreover, E2 (B. asiatica) dissolved only 5.4 mg of bilirubinate pre- dominant black PGS which was almost half of the dissolution obtained for CCGS. Form all of these interpretations we can conclude that E2 (B. asi- atica) is effective to dissolve cholesterol containing gallstones like CCGS. E4 (C. europaea), although, was found to be more efficient to dissolve black PGS than CCGS, there is no significant difference between the val- ues obtained (8.5 mg of black PGS vs 7.3 mg of CCGS). Dissolution of black PGS in E4 (C. eu- ropaea) is far close to the dissolution in E5 (T. of- ficinale) with only 1.1 mg difference. On the other hand, a significant difference was obtained for dis- solution of CCGS in E4 (C. europaea) and the most efficient plant E2 (B. asiatica) and positive control EtOH (difference of 3.5 mg with E2 and 7.0 mg with EtOH). Therefore, it can safely be inferred that E4 (C. europaea) was also effective in dissolving pig- ment stones like GSB. Among different medicine extract, M3 (Gok- shuradi) was the most effective to dissolve CCGS, it dissolved CCGS (5.8 mg) more efficiently than black GSB (3.5 mg, 16.20%). Even though, M3 (Gokshuradi) including other medicine extracts were far less effective than E2 (B. asiatica) (10.8 mg) and E4 (C. europaea) (7.3 mg) for dissolv- ing CCGS. On the other hand, among different medicines, M2 (Calcury) showed the highest dis- solution for black PGS (4.0 mg). But, M3 was still less effective than E1 (M. uniflorum), E2 (B. asi- atica), E4 (C. europaea), and E5 (T. officinale). Therefore, it can be inferred that the medicine ex- tracts were less effective than most of the plant extracts in dissolving gallstones. These ayurvedic medicines are recommended for oral dissolution therapy of kidney stones and not for gallstones. This fact could explain lesser efficacy of these com- mercial ayurvedic medicines over plant extracts in dissolving gallstones. Chekroune and Benamara [30] reported dif- Bijaya B.K. et al./ BIBECHANA 20 (2023) 175-182 180 ferent plant preparations to dissolve a significant amount of GS. About 209.37 mg, 97.42 mg, and 15.02 mg of the cholesterol-bilirubin containing GS were reduced in Herniaria hirsuta extract, lemon juice, and their mixture respectively after 312 hours of immersion whereas the olive oil/lemon juice emulsion dissolved the stone with 291.1 mg weight completely after 168 hours of immersion [30]. In the present study, among all plant extracts and gall- stone samples, the maximum weight reduction after 190 h was 10.8 mg in E2 (B. asiatica) for CCGS. Due to the difference in immersion period of the stone, direct comparison of our report with the re- ported data seems to be inappropriate, however, it can be concluded that we also found in vitro cholelitholytic activity of different plant extracts. The in vitro dissolution study showed that CCGS was more soluble in EtOH than in EDTA whereas black PGS is more soluble in EDTA than in EtOH. The insoluble residue of CCGS was left even after 190 hours of incubation in EDTA (5.1 mg dissolution) whereas CCGS was completely dis- solved in EtOH after 94 hours (11.6 mg dissolu- tion). The efficacy of EDTA to dissolve black PGS (11.5 mg) was almost double that for CCGS (5.1 mg); however, almost half of the black PGS was left undissolved even after 190 hours of incuba- tion (Annexure 1: Table 3). This indicates that CCGS is easily dissolvable by using cholesterol sol- vent like ethanol whereas black PGS contains a greater proportion of insoluble components and it is difficult to dissolve completely both in cholesterol solvent like EtOH and in calcium chelating solvents like EDTA. This finding is consistent with the result reported by Lin et al. [24]. Lee and co-worker [31] reported that 1.1 mg, 9.1 mg, and 30.0 mg of the CGS stone sample with 69.3 mg initial weight was dissolved in absolute ethanol after 3, 6, and 9 hours of incubation, respectively and the stone was dissolved completely after 18 h. In the present study, we have recorded 2.7 mg and 14.3 mg dissolution of the CCGS (wt. 14.48±0.21 mg) after 4 and 94 hours respectively. The stone was dissolved completely after 94 hours. Based on this data, the dissolution we obtained in EtOH for CCGS was lower than that reported by Lee et al. The difference arises due to the lower concentration of EtOH we used (95% EtOH). In the case of black PGS (wt. 21.46±0.22 mg), EtOH was not found effective and only 8.6 mg of the stone was dissolved after 190 h. The low solubility of the black PGS in EtOH is due to the presence of a high concen- tration of insoluble bilirubinate as the main com- ponent which was confirmed by SEM and EDS in a separate study by our group [28]. 5 Conclusion From the morphological and cross-sectional study, gallstones were classified as combined cholesterol gallstone (CCGS) or black pigment gallstone (black PGS). In vitro dissolution studies were also con- ducted using plant and medicine extracts, revealing that T. officinale was the most effective in dissolv- ing black PGS, whereas B. asiatica was the best for CCGS. M. uniflorum and C. europaea were also found to be effective in dissolving black PGS, while K. pinnata was the least effective for both types of gallstones. B. ciliate and C. europaea were equally effective in dissolving both types of GS. Medicine extracts were less effective than plant extracts. This in vitro study showed the plants can dissolve GS ef- fectively. However, the effectiveness of the plant to dissolve GS depends on the type of the stone. The study suggests that further research is necessary to confirm the effectiveness of these plants in real bi- ological systems through animal models. Further- more, the potential compound in plant that show cholelitholytic or anti-cholelithogenic activity and mechanism of action is still unknown and requires further research. The present research is limited to in vitro study. Although normal human body tem- perature (37 ℃) was maintained while preforming in vitro dissolution study, other factors like normal bile pH is not considered. List of abbreviations GS = Gallstone CCGS = Combination cholesterol gallstone Black PGS = Black pigment gallstone CDCA: Chenodeoxycholic acid UDCA: Ursodeoxycholic acid MTBE: Methyl tert-butyl ether MMP: 2-Methoxy-6-methylpyridine EDTA: Ethylenediaminetetraacetic acid Ethics approval and consent to participate Not applicable. Human and animals right No animals/humans were used for studies that are basis of this research. Availability of data and materials All the data are provided in the Supplementary ma- terial which is available on the publisher’s website along with the published article. Bijaya B.K. et al./ BIBECHANA 20 (2023) 175-182 181 Conflict of interest The authors declare no conflict of interest, financial or otherwise. Acknowledgements We acknowledge the National Youth Council, Gov- ernment of Nepal, for providing a partial research grant to conduct this research. 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Introduction Materials and Methods Collection and Pre-treatment of Gallstones Macroscopic Classification of Gallstones Collection of Plant and Ayurvedic Medicines In vitro Dissolution Study of Gallstones Dissolution of Gallstones with Plant and Medicine Extracts Results General Observation and Macroscopic Classification of Gallstones In vitro dissolution study of gallstones Discussion Conclusion