Antidiabetes constituents, cycloartenol and 24-methylenecycloartanol, from Ficus krishnae

Ficus krishnae stem bark and leaves are used for diabetes treatment in traditional medicines. Stem bark of F. krishnae was sequentially extracted with hexane, methanol and water, and these extracts were tested for their antihyperglyceamic activity by oral glucose tolerance test (OGTT) in overnight fasted glucose loaded normal rats. Hexane extract showed significant glucose lowering activity in OGTT, and the triterpene alcohols (cycloartenol+24-methylenecycloartanol) (CA+24-MCA) were isolated together from it by activity guided isolation and characterized by NMR and mass spectroscopy. The ratio of the chemical constituents CA and 24-MCA in (CA+24-MCA) was determined as 2.27:1.00 by chemical derivatization and gas chromatographic quantification. (CA+24-MCA) in high fat diet-streptozotocin induced type II diabetic rats showed significant antidiabetes activity at 1 mg/kg and ameliorated derailed blood glucose and other serum biochemical parameters. Cytoprotective activity of (CA+24-MCA) from glucose toxicity was evaluated in cultured RIN-5F cells by MTT assay and fluorescent microscopy. (CA+24-MCA) in in vitro studies showed enhanced cell viability in RIN-5F cells and significant protection of beta cells from glucose toxicity. Both in in vivo and in vitro studies (CA+24-MCA) showed enhancement in insulin release from the beta cells. In short term toxicity studies in mice (CA+24-MCA) did not show any conspicuous toxic symptoms. The combination of the phytosterols (CA+24-MCA) obtained through activity guided isolation of the stem bark of F. krishnae showed significant activity, and therefore is a promising candidate for new generation antidiabetes drug development.


Introduction
Diabetes is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action or both. The chronic hyperglycemia of diabetes is associated with long term damage, dysfunction and failure of different organs, especially the eyes, kidneys, nerves, heart and blood vessels [1]. The  no. 1 filter paper. Residual stem bark powder was dried solvent free and extracted with water (2 L) for 24 h on a temperature controlled magnetic stirrer (C-MAG-HS7, IKA, Germany) (300 rpm, 35˚C) and the extract was filtered through Whatman no. 1 filter paper. Hexane and methanol extracts were concentrated using a rotary evaporator (R-210, Buchi, Switzerland) at 40˚C under reduced pressure and the water extract was lyophilized (DPG001, Lyolab, India), to yield 12.0 g (2.4%, dr. wt., stem bark powder) hexane (FKEH), 59.0 g (11.8%, dr. wt.) methanol (FKEM) and 24.0 g (4.8%, dr. wt.) (FKEW) water extracts, respectively (S1 Scheme). These extracts were used for oral glucose tolerance test in overnight fasted glucose loaded normal rats.

Isolation of active compounds
FKEHF4 showed most significant glucose lowering activity (at 25 mg/kg body weight) in oral glucose tolerance test. In TLC/HPTLC (CAMAG, Switzerland) in 1.5:8.5 hexane-ethyl acetate, FKEHF4 showed a major compound and a minor compound, and it was subjected to column chromatography for further purification. Column was packed with 60 g silica gel (100-200 mesh size) in hexane, FKEHF4 (2 g) was adsorbed in 6 g silica gel (100-200), loaded over the packed column and eluted with hexane and ethyl acetate (100:0 to 75:25) into six fractions (100 ml each). First three fractions (100% hexane, 95% and 90% hexane in ethyl acetate) were pooled together based on their TLC profiles, to yield only the minor compound. Last three fractions (85%, 80% and 75% hexane in ethyl acetate) were pooled together (based on their TLC profiles) to yield the major compound. Pooled fractions were concentrated on a rotary evaporator at 40˚C under reduced pressure. Yields of minor and major compounds were 0.40 g (20%) and 1.60 g (80%), respectively (S1 Scheme). Both minor and major compounds were tested for their glucose lowering activity by oral glucose tolerance test in overnight fasted glucose loaded normal rats, and the major compound showed significant antihyperglycaemic activity.
standard (2 mg) was also derivatized using the same protocol. TLC/HPTLC profiling showed complete conversion of (CA+24-MCA) and standard CA into their trimethylsilyl derivatives. Derivatives of (CA+24-MCA) and standard CA were dissolved in 1.5 mL chloroform and 1 μl each were injected (splitless) on a Gas Chromatograph with AOC-20i autoinjector and FID (GC-2010 Plus, Shimadzu, Japan), fitted with a Rxi-5 Sil MS capillary column (5% phenyl 95% dimethyl polysiloxane, non-polar, 30 m x 0.25 mm i.d., 0.25 μm film thickness (Restek, USA). GC operation conditions: injector temperature 270˚C; GC oven was initially maintained at 50˚C (2 min), oven temperature programme: 50-280˚C (10˚C/min), hold time at 280˚C (30 min) and FID was set at 280˚C. Carrier gas nitrogen, flow rate 1.53 mL/min. GC analyses of samples were repeated four times each and relative percentages of individual components (derivatives of CA, 24-MCA) were calculated from the peak area-percent report of the GC-FID data.

Oral glucose tolerance test
Oral glucose tolerance test was performed as described elsewhere [16]. Briefly, required number of overnight fasted normal Wistar rats were selected and segregated into required numbers of groups containing six animals each. Animals in all groups received vehicle (1%

High fat diet fed-streptozotocin (HFD-STZ) induced type II diabetic rats
Male Wistar rats of 175-200 g body weight were selected and fed with high fat diet (HFD, 24% fat, 24% protein, 41% carbohydrate and 11% normal feed powder). After two weeks of HFD diet, animals were injected intraperitoneally with a single dose (35 mg/kg) of streptozotocin (STZ) in citrate buffer (500 μl/animal). Seven days after STZ injection blood samples were collected from the tail vein, blood glucose levels were determined using a glucometer and serum insulin levels were determined by ultrasensitive Rat Insulin ELISA assay kit (80-INSRTU-E01, ALPCO Diagnostics Inc., USA) [17].

Antidiabetes activity of (CA+24-MCA) in HFD-STZ type II diabetic rats
HFD-STZ induced type II diabetic rats (eighteen) with average glucose value of 270-286 mg/ dl and serum insulin values of 0.30-0.32 ng/ml were selected and grouped into three groups of six animals each. One group was kept as diabetic control (received 1% tween-80), and animals in test group received 1 mg/kg (CA+24-MCA) (dose was determined by the screening test), and the last group of diabetic animals received 500 μg/kg of glibenclamide. Along with the diabetic animals, two groups of age and sex matched male Wistar rats were selected, of which one group of six animals fed with normal pellet diet with average blood glucose value of 86.5 ± 3.2 mg/dl and serum insulin values of 0.22 ± 0.02 ng/ml was kept as normal control animals (received 1% tween-80) and other group of six high fat diet (HFD) fed animals with average blood glucose value of 121.3 ± 4.1 mg/dl and serum insulin values of 0.28 ± 0.04 ng/ml were kept as HFD control animals (received 1% tween-80). HFD control and HFD-STZ diabetic animals were fed with HFD and normal control animals were fed with normal pellet diet for entire experimental period. All animals received a daily single dose (1 ml/animal) of vehicle or drug or standard per orally for 25 days. Blood samples were collected from tail vein of all animals on 1, 5, 10, 15, 20 and 25th day and blood glucose levels were determined using a glucometer. On 25th day after blood glucose determination, all animals were sacrificed, blood serum and liver samples were collected and serum biochemical parameters and liver glycogen levels were estimated.

Cytoprotective effect of (CA+24-MCA) from gluco-toxicity in RIN-5F cells (MTT assay)
RIN-5F cells were cultured in RPMI media supplemented with 10% FCS, 100 μg/ml streptomycin and 100 units/ml penicillin in a carbon dioxide incubator at 37˚C, 5% carbon dioxide and 95% air for 48 h. The cultured cells were trypsinised and adjusted the cell number to 1x10 5 cells/ml with complete media containing normal glucose (5.5 mM) and other set of cells in complete media with high glucose (25 mM). Gluco-toxicity on RIN-5F cells was determined by MTT assay. Precisely, cells in both normal and high glucose medium (1x10 5 cells/ml) were cultured in 24 well plates for 24 h in the carbon dioxide incubator. In case of normal glucose medium, the cells in first three wells were kept as control incubated with 0.1% DMSO, the cells in second, third and fourth sets of wells (triplicate) were incubated with 0.1 μg/ml, 1 μg/ml and 10 μg/ml of (CA+24-MCA) for 48 h. After 48 h of incubation the spent medium was replaced with fresh medium containing 20 μl of 5 mg/ml of MTT and incubated for 4 more h in the carbon dioxide incubator. After incubation, the cells were harvested and added with 1 ml of DMSO and optical density was determined at 570 nm in plate reader (iMARK plate reader, Bio-Rad, USA). Based on the optical density the cytoprotective activity (cell proliferation) of (CA+24-MCA) was calculated. In order to determine the cytoprotective activity of (CA +24-MCA) on RIN-5F cells in high glucose medium, similar MTT assay was performed wherein instead of low glucose media, complete media with high glucose was used.

Cytoprotective effect of (CA+24-MCA) from gluco-toxicity in RIN-5F cells (fluorescent microscopy)
In order to determine the cytoprotective effect of (CA+24-MCA) in RIN-5F cells cultured in normal and high glucose media, 1x10 5 cells/ml were cultured in RPMI media containing 10% FCS, 100 μg/ml streptomycin and 100 units/ml penicillin with normal (5.5 mM) or high glucose (25 mM) for 24 h. RIN-5F cells in normal or high glucose medium were incubated with 0.1% DMSO, 0.1 μg/ml, 1 μg/ml or 10 μg/ml of (CA+24-MCA) for 48 h at 37˚C, 5% carbon dioxide and 95% air. After 48 h of incubation, spent medium was removed, cells were stained with 50 μl of acridine orange-ethydium bromide (AO-EtBr) stain (100 μg/ml) and observed under an inverted microscope (CKX41, Olympus, USA) with blue filter and photographed with a Zoom digital compact camera (CAMEDIA C-4000, Olympus, USA). Viable live cells and dead non-viable cells appeared in green and orange-red colour, respectively.

Insulin release from RIN-5F cells by (CA+24-MCA)
Insulin release from RIN-5F cells was determined as described in Ajikumaran et al., 2014 [6]. Briefly, RIN-5F cells (1x10 5 cells/ml) were cultured in 24 well plates with RPMI medium containing 10% FCS, 100 μg/ml streptomycin and 100 units/ml penicillin with normal (5.5 mM) or high glucose (25 mM) for 48 h at 37˚C and 5% CO 2 . Control and tests were done in triplicate. After incubation, spent medium was removed and cells were washed thrice with 1 ml of glucose free Krebs-Ringer bicarbonate (KRB) buffer, pH 7.4. These cells were incubated with 1 ml of glucose free KRB buffer supplemented with 1 mg/ml BSA, 10 mM HEPES for 2 h. After incubation the medium was changed and incubated with 1 ml of fresh glucose free KRB medium with 0.1% DMSO, or 0.1 μg/ml or 1 μg/ml or 10 μg/ml of (CA+24-MCA) for 1 h in CO 2 incubator at 37˚C. The supernatants were separated and insulin contents were determined with ultrasensitive Rat Insulin ELISA assay kit following the manufacture's instruction (80-INSRTU-E01, ALPCO Diagnostics Inc., USA) using a plate reader (iMARK Plate Reader, Bio-Rad, USA).

Subacute toxicity of (CA+24-MCA) in mice
Subacute toxicity evaluation of (CA+24-MCA) was carried as described in Shylesh et al., 2005 [18]. Briefly, twenty four male Swiss Albino mice (25-28 g body weight) were divided into four groups of six animals each. One group was kept as control received 1% tween-80 and second, third and fourth group of animals received 15 mg/kg, 75 mg/kg or 150 mg/kg of (CA+24-MCA), respectively, for 30 days (1 ml daily per orally). Food and water intake and behavior of animals were observed daily for 1 h for 29 days. Animals were sacrificed on the 30th day, important organs were dissected out and observed for pathological and morphological changes, blood samples were collected to determine the hematological and serum biochemical parameters.

Hematological and serum biochemical parameters, liver glycogen
Hemoglobin was measured using hemoglobinometer with comparison standards. Glutamate pyruvate transaminase (GPT), glutamate oxaloacetate transaminase (GOT) and alkaline phosphatase (AP) were measured by standard methods [19]. Urea, cholesterol, triglycerides, creatinine and protein were determined by conventional methods [20]. Peritoneal macrophages and total leucocytes were collected and counted as described by Babu et al., 2003 [21]. Serum glucose was estimated spectrophotometrically using a commercial assay kit (Monozyme, India, Ltd). Liver glycogen was estimated by the method of Carrol et al., 1956 [22]. Serum insulin was determined with ultrasensitive rat insulin ELISA assay kit (80-INSRTU-E01, ALPCO Diagnostics Inc., USA).

Statistical analysis
Statistical comparisons were done using one-way ANOVA followed by Dunnett's post hoc comparisons and student's t-test was performed in paired data analysis. P values < 0.05 were considered to be significant.
Among the seven fractions of FKEH (FKEHF1 to FKEHF7), FKEHF1, FKEHF6 and FKEHF7 with yields < 5% were subjected to oral glucose tolerance tests at a higher dose of 5 mg/kg, and the major fractions (FKEHF2, FKEHF3, FKEHF4, FKEHF5) with yields > 5% were tested in the range of 20-30 mg/kg. FKEHF4 at 25 mg/kg dose showed most significant glucose lowering activity at 30, 90, 150 min after glucose administration, and the values were 106.5 ± 2.9, 93.3 ± 2.5, 77.7 ± 2.6 mg/dl, respectively, compared to the control values of 132.5 ± 4.5, 121.2 ± 4.2, 95.6 ± 3.2 mg/dl, respectively (Fig 3). On chemical profiling of fractions (FKEHF1 to FKEHF7) by TLC/HPTLC, the active component (CA+24-MCA) was found only in FKEHF4, and its overlap in other fractions was not observed. FKEHF3 and FKEHF5 showed less significant glucose lowering activity compared to the control values, whereas FKEHF1, FKEHF2, FKEHF6, and FKEHF7 didn't show any significant glucose lowering activity in the glucose tolerant test.
Antidiabetes activity of (CA+24-MCA) in HFD-STZ type II diabetic rats (CA+24-MCA) at 1 mg/kg in 25 days treatment in HFD-STZ diabetic rats showed significant antidiabetes activity (Table 1), and significantly normalised derailed blood glucose levels from 348.4 ± 6.8 mg/dl (control diabetic rats) to 153.7± 2.5 mg/dl. The reduction in blood glucose levels in glibenclamide treated diabetic animals was comparable to that of the test compound. The drug treatment normalized the deranged liver weight and liver glycogen to normal values, and was comparable to the standard drug glibenclamide ( Table 2). Serum biochemical parameters of the experiments are given in the Table 3, (CA+24-MCA) and glibenclamide ameliorated the derailed serum biochemical parameters to normal values. The untreated diabetic control animals showed significant increase in GOT, GPT, urea, triglycerides, total cholesterol, creatinine, albumin and a decrease in protein levels. HFD-STZ diabetic control animals showed an increase in insulin release till the 15th day followed by a decline on 25th day, and their values on 1st, 5th, 10th, 15th, 20th and 25th days were 0.42 ± 0.06, 0.45 ± 0.04, 0.47 ± 0.06, 0.49 ± 0.05, 0.38 ± 0.08 and 0.19 ± 0.1 ng/ml, respectively. Serum insulin levels in experimental animals on 25th day are given in Fig 6. The diabetic animals on treatment with test drug or glibenclamide normalized the deranged insulin levels to normal control values.    �� P < 0.001 (compared to diabetic control); HFD-STZ, high fat diet-streptozotocin; Blood was collected 1 h after drug administration, except on day 1 (blood was collected just before drug administration).

Cytoprotective effect of (CA+24-MCA) from glucotoxicity in RIN-5F cell by MTT assay
Cells cultured in normal glucose medium showed increase in cell proliferation dose dependently and percentage increment in cell growth at 0.1 μg/ml, 1 μg/ml, 10 μg/ml doses of (CA +24-MCA) were 1.2%, 17.1% and 23.5%, respectively, compared to control cells. Control RIN-5F cells cultured in high glucose medium showed gluco-toxicity and it showed 40% reduction in cell proliferation than the control cells in normo-glyceamic medium. (CA+24-MCA) treated cells (0.1 μg/ml, 1 μg/ml, 10 μg/ml doses) in high glucose medium showed significant protection from gluco-toxicity dose dependently and the percentage(s) of cell growth were 23.6%, 92.5% and 115% respectively to that of control cells in high glucose medium. The cell proliferation in 0.1% DMSO, 0.1 μg/ml, 1 μg/ml and 10 μg/ml doses of (CA+24-MCA) in high glucose medium was compared with the cell proliferation in same drug concentration in 5.5 mM Table 3. Effect of (CA+24-MCA) isolated from hexane extract of F. krishnae on serum biochemical parameters in high fat-diet streptozotocin type II diabetic rats.  glucose media (pair wise). Control RIN-5F cells and 0.1 μg/ml (CA+24-MCA) treated cells in high glucose medium showed reduction in cell growth of 40 and 25.8%, respectively, than control cells in normal glucose medium. In higher doses (1 μg/ml and 10 μg/ml) of (CA+24-MCA)-treated cells no significant difference in cell growth was observed between higher and normal glucose medium in respective doses (Fig 7).

Cytoprotective effect of (CA+24-MCA) from glucotoxicity in RIN-5F cell by fluorescent microscopy
In fluorescent microscopy of RIN-5F cells cultured in normal glucose (5.5 mM) medium treated with 0.1% DMSO, 0.1 μg/ml, 1 μg/ml or 10 μg/ml of (CA+24-MCA) for 48 h, the control (0.1% DMSO treated) cells didn't show any significant loss of cell viability and only limited cell death was observed (Fig 8A). (CA+24-MCA) (0.1 μg/ml, 1 μg/ml or 10 μg/ml)-treated cells didn't show any cytotoxicity but increase in cell proliferation was observed dose dependently (all cells appeared in green colour) (Fig 8B-8D). RIN-5F cells when cultured in high glucose (25 mM) medium and treated with 0.1% DMSO, 0.1 μg/ml, 1 μg/ml or 10 μg/ml of (CA +24-MCA) for 48 h, the control cells (treated with 0.1% DMSO) showed significant cell death due to the high glucose induced toxicity; majority of cells were dead and appeared in red colour and limited number of cells were viable (green colour, Fig 8E). (CA+24-MCA) 0.1 μg/ml, 1 μg/ml or 10 μg/ml-treated cells in high glucose medium showed a concentration-dependent cytoprotection in RIN-5F cells from high glucose toxicity. (CA+24-MCA) 0.1 μg/ml-treated cells showed increment in number of viable cells (green colour) with limited dead cells (red colour, Fig 8F). (CA+24-MCA) 1 μg/ml or 10 μg/ml-treated cells showed significant increase in cytoprotection from high glucose toxicity (Fig 8G and 8H), and significant increase in cell viability was also observed. In comparison, the morphology of cells in panels G and H vary when compared with E (control) and F (0.1 μg/ml, CA+24-MCA). In (E and F), cells (mostly dead cells) were enlarged (and round) due to high glucose induced toxicity and absence of cytoplasmic processes. In 1 μg/ml and 10 μg/ml CA+24-MCA-treated cells in high glucose medium (G, H), cells were protected from glucose toxicity and they retained their normal morphology as in normal glucose medium. Cells which appeared in green colour were comparable to the cell viability of RIN-5F cells cultured in normoglyceamic medium at the same concentrations.

Subacute toxicity evaluation of (CA+24-MCA) in mice
In short term toxicity evaluation, 15, 75 and 150 mg/kg of (CA+24-MCA) didn't show any conspicuous toxicity in mice (Table 4). Serum ALP, creatinine, protein and triglyceride values were unaltered even at the highest dose tested (150 mg/kg) compared to those of control animals. Serum glucose and cholesterol values showed a significant reduction in all three doses tested (15,75 and 150 mg/kg), while serum GPT, GOT and urea values showed a significant

PLOS ONE
reduction only in 75 and 150 mg/kg body weight. Hemoglobin content, peritoneal macrophages, WBC count, food and water intake and body weight in all three doses of (CA+24-MCA) didn't show any significant deviation from the control values. Control and test animals exhibited no behavioral changes and their internal organs were devoid of any significant pathological and morphological changes.

Discussion
F. krishnae, though traditionally known, is least studied at molecular level for its antidiabetic activity, and thus we followed activity guided isolation to obtain its bioactive molecule(s) and pursued their mechanism of action. FKEH, FKEHF4 and (CA+24-MCA) showed significant glucose lowering activity in OGTT. Both CA and 24-MCA belong to the group of 4,4-dimethyl phytosterols (triterpene alcohols), with two methyl groups at C4 of the aliphatic A-ring. In several studies, these two triterpene alcohols were isolated together due to their more or less similar structural features (with only minor differences at the tail ends of their side chain carbons) [27][28][29]. CA and 24-MCA were also found together in many vegetable oils [30,31]. One example is, CA and 24-MCA are the major triterpene alcohols in rice bran oils [32]. Moreover, CA is a major precursor molecule for the biosynthesis of various phytosterols [33][34][35][36].
In HFD-STZ diabetes rats, (CA+24-MCA) significantly corrected the deranged blood glucose values. Liver weight, liver glycogen content and serum biochemical parameters are significant in the progression and pathogenesis of diabetes, and here in drug (CA+24-MCA)-treated diabetic animals, these deranged values were normalized. In case of high fat diet control animals, serum insulin level appeared to be high, and this is because beta cells were signaled to release higher concentration of insulin necessary to meet the metabolic requirements initiated by the high fat diet. Even though the insulin level was higher on 15th day in HFD-STZ diabetic control animals, the blood glucose level was not lowered, and this is due to the insulin resistance associated with type II diabetes. Due to the partial destruction of pancreatic beta cell mass and deranged glucose metabolism, type II diabetes progresses from insulin resistance to insulin insufficiency [17,39,40]. In this study, (CA+24-MCA) ameliorated the altered insulin resistance and normalized serum insulin level in diabetic animals. It may be due to the cumulative effect of the drug in pancreatic beta cell protection from glucotoxicity and enhancement in the growth and differentiation of beta cells population. Besides, the drug (CA+24-MCA)treatment normalized serum GOT, GPT, urea, triglycerides, total cholesterol, creatinin, albumin and protein values of diabetic animals to normal levels. This correction was either through proper insulin release by the drug and normalization of glucose metabolism and/or through the inhibition of gluconeogenesis from liver glycogen and muscle proteins.
Insulin resistance seen associated with type II diabetic subjects influences the up regulation of cholesterol biosynthesis. CA is known to have hypolipedimic activity in diabetic rats [31]. Another target of phytosterols in lipid homeostasis is apolipoprotein (apo A-I), which is the major protein involved in the transportation and metabolism of lipids and is an integral component in high-density lipoprotein (HDL). CA along with β-sitosterol is known to effectively correct the decrease in apo A-1 levels in cholesterol fed Wistar rats (compared to the treatment of β-sitosterol only) [41]. In another study in high fat diet fed C57BL/6J mice, triterpene alcohol and sterol preparation (TASP) from rice bran lowered the diet induced glucose-dependent insulinotropic peptide, up regulated the gene expression of fatty acid oxidation-related gene and down regulated the fatty acid synthesis related gene. Daisuke and co-workers showed that CA and 24-MCA cause less body weight gain, promote fat utilization, enhance the expression of genes associated with lipid oxidation and suppress the gene expression related to lipid biosynthesis. Besides, CA and 24-MCA specifically lowered the gene expression of sterol regulatory element-binding protein (SREBP)-1c. By these actions, the triterpene alcohols and sterols prevented diet induced obesity [42,43]. Shimotoyodome and co-workers recently showed that the consumption of brown rice, with enriched contents of CA and 24-MCA, led to a lower postprandial hyperglycemia [44,45]. Further, Tanaka and co-workers demonstrated the antihyperglyceamic effects of cycloartanol and 24-MCA along with lophenol, 24-methyl-lophenol, 24-ethyl-lophenol isolated from Aloe vera gel in type II diabetic BKS.Cg-m(+/+)Lepr(db/J) (db/db) mice [46]. The secondary starvation which existed in diabetic scenario enhanced protein degradation and deranged oxidative deamination of amino acids leading to the formation of excess ammonia culminating in the formation of higher levels of serum urea. In later stages, the consistently elevated urea level in blood leads to the dysfunctioning of kidneys, resulting in deranged albumin and creatinin levels. These conditions were abolished by the drug (CA +24-MCA) in diabetic animals, which was reflected in the normalization of the serum urea, albumin, protein and creatinin values. In addition to the antidiabetes activity, CA possessed a broad range of pharmacological activities such as anti-inflammatory, antitumor, antioxidant, antibiosis and anti-Alzheimer's disease [47].
In our study, in normoglyceamic medium, 0.1, 1.0 and 10 μg/ml doses of (CA+24-MCA) showed concentration dependent increment in RIN-5F cell proliferation (in MTT assay). Previous studies showed glucotoxicity, due to high levels of glucose in blood serum, as a major cause in the pathogenesis of diabetes [48]. RIN-5F control cells in high glucose medium showed low insulin secretion. One of the reasons for this reduction in insulin secretion of control RIN-5F cells in high glucose medium is the lower number of viable cells due to glucotoxicity. At the same time, the inherent capacity of remaining cells in control group to release basal insulin level in high glucose condition is the reason for normalization of insulin release, but it is still less than the insulin release from RIN-5F cell in normal glucose medium. In RIN-5F cells grown in high glucose medium, the drug showed dose dependent protection from glucotoxicity and significant increment in cell growth. These results are in correlation with the data obtained from the fluorescent microscopic images. In normal and high glucose medium, the enhanced secretion of insulin by RIN-5F cells compared to control cells is due to the insulin secretagogue activity of (CA+24-MCA). In a nutshell, the increment in insulin release is due to the cumulative effect of the drug on cytoprotection of RIN-5F cells from glucotoxicity, beta cell proliferation and insulin secretagogue activity of (CA+24-MCA). Cytoprotection and beta cell proliferation are important targets for the treatment of type II diabetes. Plant derived compounds (including terpenoids) are reported to show beta cell protection from hyperglycemia and amelioration of diabetic complications [38,[49][50][51][52].
In short term toxicity evaluation in mice, (CA+24-MCA) didn't show any conspicuous toxic symptoms even at 150 times higher concentration of the therapeutic dose. Body weight, food and water intake and behavioral activities of the animals were normal. Serum hematological and biochemical parameters were normal and the drug at 15, 75 and 150 mg/kg showed a significant reduction in glucose, cholesterol, urea and SGOT values. Though the drug (CA +24-MCA) has glucose lowering action, it didn't exert any hypoglycemic shock even at higher doses and maintained the glucose level close to normal control values. The reduction in serum cholesterol, urea and GOT values are additive qualities of the drug, and due to these effects diabetic complications such as hypercholestremia, nephropathy, cardiovascular issues and liver damage can be ameliorated by the drug. Briefly, in this study, we validated the antidiabetes activity of F. krishnae and isolated its antidiabetes compounds, (CA+24-MCA).

Conclusions
F. krishnae is a new source of the two phytosterols (CA+24-MCA), which exert antidiabetes activity by protecting beta cells from glucotoxicity associated with diabetes, enhancing beta cell population and normalizing insulin release from pancreatic beta cells. These terpenoids (CA +24-MCA) also have the potency to ameliorate the altered serum biochemical parameters in diabetic animals to normal level. In short term toxicity evaluation (CA+24-MCA) is devoid of any conspicuous toxic symptoms even at 150 times higher dose of the therapeutic dose, which is an additive quality of the drug. So, (CA+24-MCA) is a promising candidate for the development of new generation antidiabetes drugs.