The effect of Macrotyloma uniflorum seed on bile lithogenicity against diet induced cholelithiasis on mice.

BACKGROUND: The seeds of Macrotyloma uniflorum Lam. (Family Fabaceae) contain extractable total and tannins with reported hepatoprotective, hypocholesterolemic and antioxidant activity. In this study, dietary M. uniflorum seed, methanolic and acetone extracts (ME and AE) were examined for their bile-antilithogenic potential.
MATERIALS AND METHODS: Mice fed with 1% cholesterol and 0.5% cholic acid lithogenic (LG) diet for 8 weeks resulted in cholesterol super saturation in gallbladder bile, which promotes the formation of cholesterol gallstones (CGSs).
RESULTS: AE reduced the CGS incidence by 60.21%, and serum total cholesterol, triglyceride (TG), very low density lipoprotein (LDL) and LDL compared to control animals. Seed extracts at 300 mg/kg dose markedly reduced biliary cholesterol (BC) and decreased bile salt content. The ratio of BC to phospholipid which was 2.64 in the LG diet group was reduced to 1.57-1.35 in the M. uniflorum seed extracts treated groups. Liver cholesterol and TG were decreased significantly by feeding of ME and AE at 300 mg/kg dose. AE significantly reversed the changes in apolipoproteins A-I and C-II level disturbed by LG diet.
CONCLUSIONS: M. uniflorum seed exerted antilithogenic influence by decreasing the cholesterol hyper-secretion into bile and increasing the bile acid output, thus decreasing the formation of LG bile in mice. The effect was maximum in the AE as it also reduced papillary proliferation of gallbladder and fatty degeneration of the liver. The potential antilithogenic effect of the AE of M. uniflorum may be due to antioxidant property of its rich total polyphenol and tannins content.

INTRODUCTION

Cholesterol gallstone (CGS) disease is a highly prevalent gastroenterological disorder resulting from an alteration in hepatic and biliary cholesterol (BC) homeostasis. CGS disease is a multifactorial disease involving both environmental and genetic factors.[12] Gallstones formed in the gallbladder consist of cholesterol, bilirubin, and calcium salts. Lithogenic (LG) bile, gallbladder stasis and short nucleation time generally precede the occurrence of CGS. Relative concentration of bile acids, phospholipids (PLs) and cholesterol determine bile lithogenicity. Disruption of cholesterol homeostasis leads to increased cholesterol secretion and subsequently to super saturation of bile with cholesterol in turn called LG bile.[3] Cholelithiasis due to altered secretion rates of biliary lipids remain unknown, it is also not clear as to whether defects exist in hepatic lipid regulatory enzymes responsible for cholesterol or in bile acid biosynthesis. Bile acids are excreted through feces and a major portion is reabsorbed in the intestine and assimilated by enterohepatic circulation. Increased biosynthesis of cholesterol and decreased excretion resulting increased secretion into bile may increase the risk of CGS incidence.[4]

Several dietary components like case in, animal protein and foods containing saturated fats favor CGS formation, whereas foods containing soya protein, dietary fiber and polyunsaturated fat such as fish oil result in the lower incidence of CGS.[567] Very little information is available on the role of spices, culinary herbs and edible pulses in CGS formation. Edible herbs and seeds are valued for their organoleptic as well as medicinal properties. Previously, it has been demonstrated that dietary curcumin and capsaicin are effective in inhibiting CGS formation and also in the regression of preformed CGS in experimental mice.[8] Since the formation of CGS in the gallbladder is preceded by super saturation of bile with cholesterol, any hypocholesterolemic substance may serve as a potential dietary adjunct to reduce the incidence of CGS. Macrotyloma uniflorum seed extract is one such substance, and hence it is very relevant to study its effect during LG diet induced CGS.

Macrotyloma uniflorum Lam. (Family Fabaceae) commonly known as Horse gram is largely cultivated, especially in dry areas of Australia, Burma, India and Sri Lanka. The plant is used as a vegetable in India, and the seed is known as the poor man's pulse in southern India. Consumption of horse gram seeds, after soaking/dry heating followed by cooking, along with cooked rice or pearl millet is common among the rural people in India.[9] According to Ayurveda-the seed is bitter, acrid, hot, dry and used as astringent, anthelmintic, antipyretic and in conditions such as uterine stones, tumors, asthma, bronchitis, hiccup, urinary discharges, heart-troubles, diseases of the brain and eyes, intestinal colic, piles, inflammation, liver troubles etc., Its decoction is used traditionally in leucorrhoea and menstrual dysfunctions.[10]

Macrotyloma uniflorum seeds contain extractable total phenolics and tannins, and dry heated samples were found to have a higher content of phenolics and tannins than in raw samples.[11] Kawsar et al.[12] isolated phenolic acids from the seeds of M. uniflorum by reversed phase HPLC. Eight phenolic acids were isolated from the ethanolic extract, and the most abundant was p-coumaric acid and p-hydroxy benzoic acid.

The methanolic extract (ME) of Dolichos biflorus (synonym of M. uniflorum) whole plant possesses antihyperlipidemic activity.[13] High-fat diet induces oxidative stress in liver cells by producing reactive oxygen species.[14] Administration of D. biflorus extract to rabbits with high-fat diet induced oxidative stress showed improvement in antioxidant enzymes such as superoxide dismutase, catalase and increased glutathione concentration.[15] Chaitanya et al.[16] reported the antiurolithiatic activity of M. uniflorum seed extract on ethylene glycol induced urolithiasis in albino rats. M. uniflorum seed showed significant hepatoprotactive properties in D-Galctosamine and paracetamol induced hepatotoxicity on rats.[17] Ravishankar and Priya[18] reported in vitro antioxidant activity of ethanolic seed extracts of M. uniflorum.

Traditional healers recommend M. uniflorum seed water infusion for patients of kidney or gall stones.[192021] M. uniflorum seed is reported to possess antioxidant, hepatoprotective and anti-hyperlipidemic activity along with anti-hypercholesterolemic effect against high-fat diet-induced hypercholesterolemia in rats.[22] All the above-mentioned effects can contribute potentially toward reduction of cholesterol secretion subsequently super saturation of bile with cholesterol by controlling hepatic lipid regulatory enzymes responsible for cholesterol biosynthesis. Even through the processed seeds are increasingly consumed as human food, the beneficial effect of their bioactive compounds remain unexplored.[23] The present study was aimed at evaluating the effect of phenolic constituents of methanol and subsequently 70% acetone extract (AE) of M. uniflorum for their anti LG property on mice. The anti LG potential of M. uniflorum extracts was evaluated against LG diet-induced CGS formation in mice.

MATERIALS AND METHODS

Plant materials

The M. uniflorum seeds were collected in the month of November-December 2010 from Godavari District, Andhra Pradesh. The plant was authenticated by Dr. Zea ul Hasan Astt. Professor, Head, Department of Botany, Safia Science Collage, and a specimen voucher no. 192/BOT/Safia/10 was preserved for future reference. Dust and debris were removed from the seeds, and they were spread out at room temperature for complete drying. Dried seeds were powdered with a domestic grinder.

Preparation of seed extracts

Powdered seed samples were defatted using petroleum ether with the ratio of sample to solvent being 1/10 w/v with occasional shaking at room temperature for 24 h. The extract was filtered through Whatman filter paper and air-dried. The residue was extracted again with methanol for 24 h, filtered and re-extracted with an additional quantity of methanol for 3 h. The combined ME were stored for further analysis.

The residue left out after air-drying was again extracted by occasional shaking with 70% acetone at room temperature for 24 h, filtered and re-extracted for 3 h. The respective combined AE were evaporated under reduced pressure using a rotary vacuum evaporator. The extracts (ME and AE) thus obtained were used for qualitative phytochemical analysis and estimation of total phenolic and tannin content.

Determination of total phenols and tannin contents

The total phenolic content of the freeze-dried ME and AE were determined according to the method described by Makkar.[24] Aliquots of the extract ME and AE 0.5 ml each (0.1 mg/ml) were taken and made up to a volume of 1 ml with distilled water. Then 0.5 ml of Folin–Ciocalteu reagent (1:1 with water) and 2.5 ml of sodium carbonate solution (20% w/v) were added sequentially in each tube. Soon after vortexing the reaction mixture, the tubes were placed in the dark for 40 min and the absorbance was recorded at 725 nm against the reagent blank with a double beam ultraviolet/visible spectrophotometer (EI model No. 1372, Japan). The amount of total phenolics was calculated as pyrogallol equivalents (even though pyrogallol was used as a standard in this work, it is not a natural constituent of horse gram or legumes in general) from the calibration curve.

The tannin content was estimated after treatment with polyvinyl polypyrrolidone (PVPP) following the method of Makkar.[24] PVPP content 100 mg/ml was taken in a test tube and to this, 1.0 ml of the ME and AE were added. The content was vortexed and kept at 4°C for 15 min, centrifuged (3000 rpm) for 10 min at room temperature, and the supernatant was collected. This supernatant had only simple phenolics other than tannins as PVPP had precipitated the tannins as residue. The phenolic content of the supernatant was measured as mentioned above and expressed as the content of nontannin phenolics on a dry weight basis. From the above results, the tannin content of the sample was calculated following the formula on dry weight basis and then to percentage yield with respect to starting material weight.

Total phenolics − Nontannin phenolics = Tannin content

Estimation of total flavonoid

Aluminum chloride colorimetric technique was used for estimation of flavonoids. Totally, 0.5 ml each of ME and AE was taken (100 mg/ml of methanol) in test tube and mixed with 1.5 ml of methanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water. The reaction mixture was left at room temperature for 30 min. and the absorbance was measured at 415 nm. The calibration curve was plotted against quercetin standard at concentration range of 10–70 ppm in methanol. The amount of flavonoid was calculated from the standard graph.[25]

Test animals and diet

Laboratory bred Wistar albino mice of both sexes (20–25 g) maintained under standard laboratory conditions at 22 ± 2°C, relative humidity 50 ± 15% and photoperiod (12 h dark and light) were used for the experiment. Commercial pellet diet (Hindustan Lever, India) and water were provided ad libitum. In order to avoid diurnal variation, all the treatments were carried out at same time of the day, that is, between 10 a.m. and 5 p.m. Approval (Institutional Animal Ethical Committee [IAEC]/RCP/OCT 2010/06) was obtained from IAEC (approved Body of Committee for the Purpose of Control and Supervision of Experiments on Animals, Chennai, India) from our institute before carrying out the experiments and care provided to the animal was as per the World Health Organization “guidelines for the care and use of animals in scientific research.”

The animals were divided randomly into control and seven experimental groups of eight mice each and fed a diet of ground pellets to which 1% cholesterol, 0.5% cholic acid (LG diet) had been added as per Scott.[26] All groups of animals were maintained in separate cages with free access to water. Control animals were fed the ground pellet diet and water ad libitum.

Determination of lethal dose, 50%

Lethal dose, 50% (LD50) was determined according to the guidelines of Organization for Economic Co-operation and Development (OECD) following the up and down method (OECD guideline No. 425) and fixed dose method (OECD guideline No. 420). Based on the results of these tests, a limit test was performed to categorize the toxicity class of the compound and then Main test was performed to estimate the exact LD50.[27] The limit test was started at a dose of 2000 mg/kg. LD50 was found greater than the test dose so the test substance dose was classified in the hazard classification as Class 5, which is nontoxic.

Study protocol

Mice were divided randomly 8 per group and into 7 groups. All the animals other than the control group were given the LG diet for 8 weeks. After 8 weeks all the other groups except the LG diet control group were given in addition, ursodiol (150 mg/kg/day suspended in 0.5 ml saline), 150 and 300 mg/kg dose of both ME and AE by oral route. The animals of normal diet control group were given an equal volume of saline. Body weight was recorded at weekly intervals. After 6 weeks of treatment, overnight fasted animals were sacrificed under anesthesia. Blood was withdrawn immediately following cardiac puncture and collected in a clean and dry test tube. The blood was allowed to coagulate for 30 min, centrifuged at 3000 rpm for 5 min and serum collected. The liver was dissected out, washed with ice-cold saline, blotted dry, weighed, and a part of the liver was preserved in cold saline for estimation of enzymes. Gallbladder was carefully collected, length measured and weighed. Bile was aspirated with a thin needle and kept for subsequent analysis. The gallbladder was cut open under microscope, and the gallstones were evaluated according to the score criteria of Juniper and Burson[28] and incidence recorded.

Analysis of serum lipid parameters

Estimation of serum total cholesterol (TC),[29] triglyceride (TG),[30] high-density lipoprotein cholesterol (HDL-c),[31] alkaline phosphatase (ALP),[32] and total bilirubin and direct bilirubin[33] was done in Span auto analyzer (India) using commercial kit (Span Diagnostics Pvt. Ltd., India). Serum low-density lipoprotein cholesterol (LDL-c) and very LDL-c (VLDL-c) content was calculated with the formula using serum concentration values of TC, TG, and HDL. Apolipoproteins (APOs) concentration in serum was measured by radial immune diffusion assay for APO A-1,[34] APO C-II and APO C-III.[35]

Analysis of liver lipid parameters

Hepatic and biliary lipids were extracted by the method of Bligh and Dyer[36] with chloroform and methanol (2:1 v/v), and the chloroform phase was used for TC and TG analysis. Bile PLs (in bile is entirely lecithin) was measured by the method of Stewart[37] using dipalmitoyl phosphatidylcholine as a reference standard.

Analysis of bile lipid parameters

Gallstones were crushed along with the bile fluid and used for lipid extraction. From the extracted bile, the chloroform phase was used for TC analysis and the upper methanolic phase was used for the enzymatic estimation of total bile salts using 3a-hydroxysteroid dehydrogenase.[38] Hepatic bile content was measured by the method of Stewart.[37]

Histopathological studies of gallbladder and liver

A part of the liver was stored in Aqua Bouine's fluid for histopathology. Gallbladder and liver slices were fixed, paraffin embedded and processed for histopathological assessment of damage following the method of Lee et al.[39]

Statistical analysis

Experimental data were analyzed using one-way ANOVA followed by Tukey-Kramer multiple comparison test. P < 0.05 was considered statistically significant. Graph Pad Prism software suite, Version 3.02 was used for statistical analysis.

RESULTS

The yield of M. uniflorum methanol extract was higher with rich presence of flavonoid (8.59 mg/g). AE showed high content of total polyphenol and tannins (8.91 and 5.23 mg/g respectively) as shown in Table 1.

Table 1

Quantitative estimation of phytoconstituents in Macrotyloma uniflorum seed

Effect on the incidence of cholesterol gallstones

The gallbladder size and weight were significantly increased by feeding of LG diet. Animals fed with the control diet showed no sign of any cholesterol crystal or stone in the gallbladder, whereas all the animals in a LG diet group showed CGS [Table 2]. Animals fed with the LG diet supplemented with M. uniflorum extracts showed a significant reduction in the incidence of CGS. The percentage incidence of CGS was calculated by taking the incidence in LG diet as 100%. Both extracts effectively reduced the formation of CGS, the lowest incidence of CGS (60.21%) and also significant (P < 0.01) reduction of gall bladder weight was observed in AE 300 mg/kg group.

Table 2

Effect of Macrotyloma uniflorum extracts on the incidence of cholesterol gallstones on LD induced experimental lithogenesis in mice

Effect on serum lipid, apolipoprotein, and biochemical parameters

The LG diet feeding induced hypercholesterolemia with approximate 2-fold increase in serum TC, TG, VLDL and LDL compared to control animals. At 300 mg/kg dose AE of M. uniflorum prevented the rise in all serum lipid parameters and ME effectively reduced TC, TG and LDL levels [Table 3].

Table 3

Effect of Macrotyloma uniflorum extracts on serum lipid parameters of LD induced experimental lithogenesis on mice

Methanoli extracts and AE at 300 mg/kg dose significantly (P < 0.001) decreased the level of ALP, total, and direct bilirubin rose by LG diet. LG diet significantly raised serum bile and APO A-I content and reduce APO C-II and C-III. AE in both the doses significantly reverted the changes in APO A-I and C-II level whereas ME was ineffective. Both extracts were unable to normalize the raised level of serum bile in the tested doses [Table 4].

Table 4

Effect of Macrotyloma uniflorum extracts on serum APO and biochemical parameters of LD induced experimental lithogenesis on mice

Effect on hepatic lipid parameters

There were no significant differences in the body weight of animals between various diet groups at the end of the 6 weeks feeding period. The liver weight was higher in the LG diet group. Bile contains PLs and bile salts, emulsifying agents that are necessary for fat absorption and digestion. Hepatic bile PL content was decreased in LG diet group compared to control animals. Feeding of LG diet increased TG content of liver and treatment of ME (300 mg/kg) significantly countered the raise in TG content [Table 5].

Table 5

Effect of Macrotyloma uniflorum extracts on hepatic lipid parameters of LD induced experimental lithogenesis on mice

Effect on biliary lipid profile

Lithogenic diet (LD) increased the cholesterol and decreased bile salt content of bile by three fold. Ursodiol (P < 0.001), ME and AE at 300 mg/kg dose (P < 0.05–0.01) countered the increase in BC and decreased bile salt content significantly. ME at 300 mg/kg dose also significantly (P < 0.05) increased bile lecithin content decreased by LG diet. The increased BC/PL ratio (2.64) resulted by the LG diet was brought down significantly (1.03) by ME 300 mg/kg dosing [Table 6].

Table 6

Effect of Macrotyloma uniflorum extracts on biliary lipid profile of LD induced experimental lithogenesis in mice

Effect on gallbladder histology

Lithogenic diet induced mucosal hyperplasia with epithelial herniation, proliferation of gland and focus sinus like invagination in mouse gallbladder. Ursodiol normalizes papillary proliferation whereas ME (300 mg/kg) treated mice gallbladder showed only papillary proliferation. AE (300 mg/kg) treatment showed low extent of papillary proliferation and metaplastic glands [Figure 1].

Figure 1

Photomicrograph of mice gallbladder section. (a) Control mice gallbladder section; (b) Lithogenic diet showing mucosal hyperplasia, epithelial herniation, fibrosis, proliferation of gland and focus sinus invagination; (c) Ursodiol treated mucosa with no papillary proliferation or elon-gation; (d) Methanol extract (ME) (150 mg/kg) showed papillary proliferation and inflammation; (e) ME (300 mg/kg) showed papillary proliferation only; (f) Acetone extract (AE) (150 mg/kg) showed hyperplastic proliferation, metaplastic glands and edema; (g) AE (300 mg/kg) low extent of papillary proliferation, metaplastic glands with epithelial herniation

Effect on liver histology

Lithogenic diet caused inflammation, necrosis, fatty degeneration, Kupffer cell nodule and erythrophagocytosis in hepatocyte. Ursodiol treatment showed normalization of fatty degeneration with only few areas of inflammation and necrosis. ME (300 mg/kg) treatment reversed fatty changes and steatosis but with inflammatory necrosis. Treatment of AE (150 and 300 mg/kg) protected the liver from fatty degeneration with only small areas of focal necrosis [Figure 2].

Figure 2

Photomicrograph of mice liver section. (a) Liver section of control mice; (b) Lithogenic diet showing small area of inflammation, necrosis, vacuolated cytoplasm, Kupffer cell nodule; (c) Ursodiol treated liver having small areas of inflammation and necrosis absence of fatty degen-eration; (d) Methanol extract (ME) (150 mg/kg) showed fatty changes, stetosis and cellular degeneration; (e) ME (300 mg/kg) showed cellular degeneration with reversal of fatty changes; (f) Acetone extract (AE) (150 mg/kg) showed reversal of fatty damage, focal inflammatory necrosis; (g) AE (300 mg/kg) showed low extent of inflammation and small focal necrosis

DISCUSSION

Formation of gallstone is a complicated pathologic process, which involves many factors. Han et al.[40] in a case–control study found that the formation of gallbladder stones is related to aging, metabolic disturbance and damage to gallbladder emptying function. To prevent the morbidity due to cholelithiasis and gallbladder stone in the young, the study of its pathogenicity is essential. In this study, the changes in plasma lipoproteins, hepatic and bile concentrations have been observed in the processes of CGS formation induced by the cholic acid-cholesterol diet in the animal model. LG diet is known to induce changes in bile components with an increase in substances such as arachidoynyl lecithin and dehydro cholesterol, which are highly damaging to gallbladder epithelial cells with membrane injury to the platelets and red cells.[41]

Lipoprotein metabolism and interaction between caloric consumption influence bile lithogenecity and gallstone formation in conjugation with dietary factors.[42] In normal people, the plasma LDL-c concentration appeared to be related to the BC concentration, but the pathway of its metabolism is still unknown.[39] In type IV hyperlipidemia patients with elevated VLDL-c and LDL-c, the incidence of gallstone was high.[42] Our study also showed significantly elevated plasma TC, TG, VLDL-c, LDL-c and lowered HDL-c in LG mice. Hayes et al.[43] found an expanded pool of VLDL-c and LDL-c and a reduced pool of HDL2 predominated in hamsters with CGSs. Likewise, increased VLDL and decreased HDL, primarily HDL2, were common in obese persons with gallstone. Therefore, elevation of VLDL-c and LDL-c is closely related to excessive dietary cholesterol.[44] Lipoprotein HDL-c are transferred to VLDL and LDL, and its decreased level bears a close relationship with elevated activity of plasma cholesteryl ester transfer protein which promotes this conversion.[2]

Robins and Brunengraber[45] suggested that the precursor cholesterol secreted by the gallbladder are transported directly from plasma to the biliary canaliculi through hepatocytes without entering the interior of the cell. The concentration of APOs in bile is about 10% of that in plasma. Although APOs are potential anti nucleating proteins in bile, their functional role in vivo as a factor in the solubilization of BC is relatively unexplored. In vitro APO A1 in low concentrations can delay the shift from micelles to vesicles, thereby enhancing the cholesterol-solubilizing capacity of bile acids. Another finding is that APO A1 stabilizes PL lamellae and thus prolongs nucleation time in model bile systems.[46] This study demonstrates that the concentration of plasma APO A1 gradually decreased following dietary cholesterol, which might result in a reduced concentration of APO A1 and cholesterol crystal formation in bile. Biliary secretion of PLs is dependent on the bile acids. During bile formation, bile acids stimulate secretion of PLs from hepatocytes. Increased secretion of cholesterol is related to PLs with more hydrophobic molecular species in the bile.[45]

Feeding of LG diet in the present study increased the hepatic cholesterol, a significant part of which was secreted into bile. Cholesterol secretion into bile is tightly coupled to decreased PL and bile acid secretion.[45] The pathophysiological conditions that might be responsible for the cholesterol crystallization and gallstone formation are increased cholesterol content and decreased bile salts in bile. It has been reported that mice susceptible to gallstones secrete more cholesterol into bile through canalicular membrane leading to the formation of LG bile.[47]

Biliary cholesterol/PL secretion rates are considered to be closely linked to bile acid secretion rates and reduced by higher bile acids in bile. The BC/PL ratio of bile indicates the ratio in the vesicles that secrets bile acids at the canaliculus.[47] In the present study, LG diet feeding significantly increased BC/PL ratio. In contrast, animals maintained on M. uniflorum extract treatment showed a significant decrease in BC/PL compared to the LG diet group. This showed that LG diet fed mice secrete more cholesterol into bile at the canalicular membrane level which is a major factor leading to the formation of LG bile. The present results are also in agreement with Hofmann et al.[48] who observed that there was a high BC/PL ratio in the bile of CGS patients. This suggests that in CGS patients as well as in mice, nascent PL vesicles at the canalicular level may be enriched with cholesterol and the test drug prevented this enrichment. Several dietary components are known to influence CGS.

Formation of CGS in the gallbladder is preceded by super saturation of bile with cholesterol mostly in animals with diets having saturated fat,[49] hypocholesterolemic foods such as M. uniflorum may serve as potential dietary adjuncts to reduce the incidence of CGS. Feeding of LG diet to mice caused secretion of more cholesterol molecules into bile leading to the formation of LG bile. Bile salts form a major part of the bile solids. They are synthesized from the parent molecule cholesterol. The majority of the cholesterol is excreted into bile after conversion into bile acids, and its increase tends to decrease the cholesterol content and in turn lithogenecity of bile.

Kern[4] reported that high dietary cholesterol increases BC secretion in the gallstone subjects, but not in normal persons, indicating that dietary cholesterol might be important in the pathogenesis of CGSs, this is also supported by the hypothesis that hepatic metabolism of cholesterol in gallstone patients differs from those without stones. Although the factors that regulate BC secretion are not certain, a number of studies have suggested that most BC is derived from the existing rather than newly synthesized cholesterol. The present study shows that dietary cholesterol increased hepatic and BC concomitantly. Lipogenicity of bile is determined by relative concentrations of three main components namely bile acids, PL, and cholesterol. Generally, LG bile occurs with disruption of cholesterol homeostasis, leading to increased cholesterol secretion and subsequent supersaturation of bile with cholesterol.[3]

Compared to the control group, the total bile acid content of bile in the LG group decreased whereas the cholesterol content increased three-fold. On the other hand, feeding of M. uniflorum significantly decreased the BC and increased the bile acids, while making the bile slightly more hydrophilic. The hydrophobicity of bile acids plays an important role in gallstone formation. With the increase in the formation of bile acids, the increased bile flow and PLs help in solubilization of cholesterol in mixed micelles and vesicles, which are the major carriers of cholesterol in bile. This causes stabilization of bile with increased solubilization of cholesterol and prolongs the cholesterol nucleation time. It has been shown that due to the concentration of bile in the gallbladder by absorption of water, elevation of biliary vesicular BC/PL ratio occurs leading to vesicular aggregation, which precedes nucleation.[50] As the bile gets concentrated in the gallbladder, the vesicles become rich in cholesterol, resulting in an increased BC/PL ratio in vesicles. With this increased ratio, the vesicles become unstable and tend to nucleate cholesterol. The lowered levels of cholesterol may be the combined result of decreased synthesis and increased conversion to bile acids due to the action of M. uniflorum. The present study clearly demonstrates that feeding of M. uniflorum inhibits the formation of CGS in mice during experimental induction by decreasing the cholesterol content in serum, liver, and bile.

Feeding of LG diet significantly increased liver weight due to high-cholesterol deposition and higher TG content. LG diet treated mice showed inflammation, necrosis, fatty degeneration and erythrophagocytosis in mice liver. M. uniflorum partially countered the increase in liver weight by reversing fatty changes by possibly correcting the alterations in the hepatic lipid profile. The study outcome shows that an LG diet can induce biliary change and resultant epithelial cell injury with stone formation. Human gallbladders with cholecystitis or cholelithiasis usually demonstrate mucosal hyperplasia with epithelial herniation into underlying walls.[51] Hyperplastic changes in the gall bladder suggest that it is a reactive process related to mucosal irritation and regeneration and has a reverse correlation with the tension on gallbladder. Gallstone formation with the resultant increase of intraluminal pressure and weakening of the wall by distention might cause inward proliferation, which was evident on LG diet treated gallbladder having extensive mucosal hyperplasia, epithelial herniation, fibrosis and proliferation of gland. M. uniflorum at 300 mg/kg oral dose showed only low extent of mucosal proliferation that signifies protection from hyperplasia with epithelial herniation.

The antioxidant activity of flavonoids and phenols are principally based on the structure relationship between different parts of their chemical structure.[52] Polyphenols are common constituents of the human diet present in most foods and beverages of plant origin. They are capable of removing free radicals, chelating metal catalysts, activating antioxidant enzymes, reducing tocopherol radicals and inhibiting oxidase.[53] Garlic (Allium sativum), fenugreek (Trigonella foenum-graecum) and onion (Allium cepa) containing phenols and flavonoids show antioxidant activity[545556] and were reported to reduce the incidence of LG diet induced CGSs in mice.[5758] Perumal and Sellamuthu[11] reported antioxidant and free radical scavenging capacity of M. uniflorum phenolic extract of raw and processed seeds having a good hydroxyl radical and DPPH* radical scavenging activity. The black variety was found to have more ferric reducing and antioxidant power than the brown variety. It contains isoflavone diglycoside, 5-hydroxy-7, 30, 40-trimethoxy-8-methylisoflavone; 5-O-α Lrhamnopyranosyl-O-β-D-glucopyranoside. The whole seeds have been reported to contain about 1.6% tannins.[59] The soup made from the seed of this plant is said to “generate heat" and help dilute renal stone due to the presence of pentacyclic triterpenes, mainly butelin, and inhibit some steps of oxalate synthesis from glycolic acid.[16] Reddy and Shrinivasan[60] reported that dietary fenugreek seeds lower the incidence of CGS in high-cholesterol fed mice and also regress pre-established CGS suggesting its hepatoprotective and antioxidant potential. The present study suggests that anti LG effect of M. uniflorum seed ME and AE may be due to antioxidant property of its rich polyphenols and tannin content.