Claims:1. A method of synthesizing an extract of Coleus forskohlii, comprises:
(a) taking a raw material;
(b) carrying out an extraction of the raw material and collecting an extract;
(c) removing oil from the extract and dissolving the extract in ethanol;
(d) removing extra oil from the extract, wherein the extra oil is removed by carrying out charcoalization of the extract;
(e) filtering the extract;
(f) concentrating the extract;
(g) crystallizing the extract, wherein crystallization is carried out at 5-10°C for 3 days;
(h) filtering the extract;
(i) performing a silica gel chromatography of the extract to separate the fraction, wherein the extract obtained is free from 14-deoxycoleon U compound that causes fatty liver in patients;
(j) drying the fraction to get C. forskohlii extract.

2. The method as claimed in claim 1, wherein the step of performing the silica gel chromatography comprises:
using a glass column packed with silica column;
loading a sample into the column;
eluting the sample with hexane at flow rate of 1ml/min;
discarding first 10 fractions and then starting a gradient elution with combination of hexane and diethyl ether;
collecting the fraction containing 14-Deoxycoleon and eluting with 100% ethyl acetate to get 14-Deoxycoloen free extract;
concentrating the extract to thick paste and adding ethanol; and
distilling out ethanol completely and drying the residue.
3. The method as claimed in claim 1, wherein the raw material is prepared by taking the roots of Coleus forskohlii plant and grinding the roots to obtain a powder.
4. The method as claimed in claim 1, wherein the extraction is carried out using an extractor and an alcohol, wherein the alcohol has a concentration of 70%.
5. The method as claimed in claim 1, wherein the oil is removed by first washing the extract with water.
6. The method as claimed in claim 1, wherein the extract free from 14-deoxycoleon U is administered to patients for treatment of obesity.
7. The method as claimed in claim 1, wherein the extract is administered in a therapeutically effective amount to a patient for promoting lean body mass in a mammal in need thereof.
8. The method as claimed in claim 1, wherein the extract prevents diet induced liver dysfunction and improves liver function by normalizing the elevated liver enzymes selected from the group consisting of SGOT, SGPT, GSH, TBR, SOD and CAD.
9. The method as claimed in claim 1, wherein the extract is free from 14-deoxycoleon U is used for treatment of inflammation in patients.
10. A pharmaceutical composition comprising an extract of Coleus forskohlii plant, wherein the composition is effective in treatment of diabetes associated with risk of hyperlipidemia and cardiovascular disease.
11. A pharmaceutical composition comprising an extract of Coleus forskohlii plant, wherein the composition activates enzyme adenylyl cyclase and increases intracellular levels of cAMP, wherein the cAMP stimulates the activity of a protein kinase which phosphorylates a hormone-sensitive lipase to produce the active form this enzyme.
12. A pharmaceutical composition comprising an extract of Coleus forskohlii plant, wherein the composition is formulated into dosage forms selected from a group comprising liquid, powder, granule, capsule, tablet, syrups, elixirs, phytoceuticals and nutraceuticals.
13. A pharmaceutical composition comprising an extract of Coleus forskohlii plant, wherein a therapeutically effective amount of the extract is between 0.01 mg and 500 mg per kilogram of body weight of an individual to which the extract is administered.
, Description:A) TECHNICAL FIELD OF INVENTION
[001] The present invention generally relates to a herbal extract and particularly to extract of coleus plant. The present invention more particularly relates to a method of synthesizing an extract of coleus.

B) BACKGROUND OF INVENTION
[002] Coleus forskohlii (CF) is a south Asian herb that has been historically used in ayurvedic medicine. The roots of Coleus forskohlii plant contains forskolin, which is a biologically active compound that has been demonstrated to improve well-being in some individuals by promoting lean body mass and supporting healthy metabolic function.Coleus forskohlii plant has been used since ancient times to treat heart disorders such as high blood pressure and chest pain (angina), as well as respiratory disorders such as asthma.
[003] Many formulations of C. forskohlii extract have been developed and are available in the state of the art. C. forskohlii extract is an important fat burner use for the treatment of obesity, liver toxicity, cardiovascular, respiratory, and central nervous system. Currently, the available solution for the treatment of fatty liver caused by fat burners is with antihepatotoxic medication. This is because of some of the phytoconstituent present in C. forskohlii extract that causes hepatotoxicity. A recent report on liver toxicity, i.e., hepatic cytochrome P450 (CYP) and fatty liver in mice showed that liver toxicity is induced by an unknown compound present in C. forskohlii extract. In addition, the study showed that the major component forskohlii exhibited zero contribution towards liver toxicity and is safe for human consumption.
[004] Hence, hepatotoxicity is caused by the consumption of C. forskohlii extract(FORCSLIM™) which contain 14-deoxycoleon U. Some in vivo studies in mice, also showed that discontinuation of Coleus forskohlii extract consumption would revert the toxic effect.In adverse condition, liver transplantation might be the only solution.
[005] The main components in C. forskohlii extract are diterpene compounds, among which 14-Deoxycoleon U is the main cause for liver toxicity. Therefore, it is likely that the unknown compound which actively induces fatty liver in C. forskohlii extract is 14-deoxycoleon U. The hepatotoxicity caused by the consumption of fat burners i.e. 14-deoxycoleon U containing Coleus forskohlii extract and the induced hepatotoxicity is usually treated with antihepatotoxic medications, which might have some side effects (documented and undocumented). The cost of hepatic treatment is generally very expensive The discontinuous use of Coleus forskohlii extract cannot be considered as a total solution because Coleus forskohlii extract forskohlii is administered as medication for other major ailments like obesity, cardiovascular disorders, adenocarcinoma etc. and treatment is usually for a particular period. Thus, discontinuous use will affect the treatment of other illnesses.
[006] Thus, there is a need to come up with a method of preparing an extract of C. forskohlii in which the compound14-deoxycoleon U is eliminated, thereby, ensuring the safety of C. forskohlii extract containing herbal dietary supplements and phytopharmaceuticals and above all healthsafety isupheld.
[007] The value additions and above-mentioned shortcomings, disadvantages and problems are addressed herein, as detailed below.

C) OBJECT OF INVENTION
[008] Thus, the primary object of the present invention is to provide a method of removing 14-Deoxycoleon U compound from the extract of C. forskohlii plant.
[009] Another object of the present invention is to provide amethod of synthesizing an extract of C. forskohlii plant with elimination of compound14-Deoxycoleon U, which is a cause of liver toxicity in patients.
[0010] Yet another object of the present invention is to provide a method of synthesizing an extract of C. forskohlii plant with eliminated 14-Deoxycoleon U compound to reduce the hepatotoxicity caused by the presence of this compound.
[0011] Yet another object of the present invention is to provide an extract of C. forskohlii free from 14-deoxycoleon U compound for use in treatment of obesity.
[0012] Yet another object of the present invention is to provide an extract of C. forskohlii free from 14-deoxycoleon U compound for promoting lean body mass in a mammal in need thereof.
[0013] Yet another object of the present invention is to provide an extract of C. forskohliifree from 14-deoxycoleon U compound which prevents diet induced liver dysfunction and improves liver function by normalizing the elevated liver enzymes selected from the group consisting of SGOT, SGPT, GSH, TBR, SOD and CAD.
[0014] Yet another object of the present invention is to provide an extract of C. forskohliifree from 14-deoxycoleon U compound for treatment of inflammatory ailments.
[0015] Yet another object of the present invention is to provide an extract of C. forskohliifree from 14-deoxycoleon U compound used in treatment of diabetes associated with risk of hyperlipidaemia and cardiovascular disease.
[0016] Yet another object of the present invention is to provide an extract of C. forskohlii free from 14-deoxycoleon U compound that activates the enzyme adenylyl cyclase and increases intracellular levels of cAMP, wherein the cAMP stimulates the activity of a protein kinase which phosphorylates a hormone-sensitive lipase to produce the active form of this enzyme.
[0017] Yet another object of the present invention is to provide an extract of C. forskohlii free from 14-deoxycoleon U compound that is formulated into dosage forms selected from a group comprising liquid, powder, granule, capsule, tablet, syrups, elixirs, phyotceuticals and neutraceuticals.
[0018] These and other objects and advantages of the embodiments herein will become readily apparent from the following detailed description taken in conjunction with the accompanying drawings.

D) SUMMARY OF INVENTION
[0019] The embodiments of the present invention provide amethod of synthesizing an extract of Coleus forskohlii, comprises:
[0020] (a) taking a raw material;
[0021] (b) carrying out an extraction of the raw material and collecting an extract;
[0022] (c) removing oil from the extract and dissolving the extract in ethanol;
[0023] (d) removing extra oil from the extract, wherein the extra oil is removed by carrying out charcoalization of the extract;
[0024] (e) filtering the extract;
[0025] (f) concentrating the extract;
[0026] (g) crystallizing the extract, wherein crystallization is carried out at 5-10°C for 3 days;
[0027] (h) filtering the extract;
[0028] (i) performing a silica gel chromatography of the extract to separate the crystals, wherein the extract obtained is free from 14-deoxycoleon U compound that causes fatty liver in patients;
[0029] (j) drying the residue to get C. forskohlii extract (1-99%).
[0030] According to an embodiment of the present invention, the step of performing the silica gel chromatography comprises using a glass column packed with silica column. The length of the silica column is 2 ft and the diameter of the silica column is 2.5 inch. The sample is loaded into the column. The sample is eluted with hexane at flow rate of 1ml/min. The first 10 fractions are discarded and then a gradient elution is started with combination of hexane and diethyl ether from (100:0) to (80:20). The fraction containing 14-Deoxycoleon is collected and eluted with 100% ethyl acetate to get 14-Deoxycoloen free extract. The extract is concentrated to thick paste and added with ethanol. The ethanol is distilled out completely and the residue is dried.
[0031] According to an embodiment of the present invention, the raw material is prepared by taking the roots of Coleus forskohlii plant and grinding the roots to obtain a powder.
[0032] According to an embodiment of the present invention, the extraction is carried out using an extractor and an alcohol, wherein the alcohol has a concentration of 70%.
[0033] According to an embodiment of the present invention, the oil is removed by first washing the extract with water.
[0034] According to an embodiment of the present invention, the extract free from 14-deoxycoleon U is administered to patients for treatment of obesity.
[0035] According to an embodiment of the present invention, the extract is administered in a therapeutically effective amount to a patient for promoting lean body mass in a mammal in need thereof.
[0036] According to an embodiment of the present invention, the extract prevents diet induced liver dysfunction and improves liver function by normalizing the elevated liver enzymes selected from the group consisting of SGOT, SGPT, GSH, TBR, SOD and CAD.
[0037] According to an embodiment of the present invention, the extract is free from 14-deoxycoleon U is used for treatment of inflammation in patients.
[0038] According to another embodiment of the present invention, apharmaceutical composition comprising an extract of Coleus forskohlii (Forcslim™) plant, wherein the composition is effective in treatment of diabetes associated with risk of hyperlipidaemia and cardiovascular disease.
[0039] According to an embodiment of the present invention, a pharmaceutical composition comprising an extract of Coleus forskohlii (Forcslim™) plant, wherein the composition activates enzyme adenylyl cyclase and increases intracellular levels of cAMP, wherein the cAMP stimulates the activity of a protein kinase which phosphorylates a hormone-sensitive lipase to produce the active form of this enzyme.
[0040] According to an embodiment of the present invention, a pharmaceutical composition comprising an extract of Coleus forskohlii plant, wherein the composition is formulated into dosage forms selected from a group comprising liquid, powder, granule, capsule, tablet, syrups, elixirs, phytoceuticals and nutraceuticals.
[0041] According to an embodiment of the present invention, a pharmaceutical composition comprising an extract of Coleus forskohlii plant wherein the therapeutically effective amount of the extract is between 0.01 mg and 500 mg per kilogram of body weight of an individual to which the extract is administered.
[0042] These and other aspects of the embodiments herein will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications.

E) BRIEF DESCRIPTION OF DRAWINGS
[0043] The other objects, features and advantages will occur to those skilled in the art from the following description of the preferred embodiment and the accompanying drawings in which:
[0044] FIG. 1 is a flow chart showing the various steps involved in the method of synthesizing an extract of Coleus forskohlii, according to an embodiment of the present invention.
[0045] FIG. 2 is a flow cytometry histogram of mean anti-inflammatory cytokines (IL-10) in THP1 cells pre-stimulated with LPS followed by 24 h exposure to FORCSLIM™, according to an embodiment of the present invention.
[0046] FIG. 3 shows the images for MTT cell viability assay at 20X, according to an embodiment of the present invention.
[0047] FIG. 4 shows the Cell viability of 3T3L1 cells treated with different concentrations of C. forskohlii (Forcslim™) extract and 100 µM of control drug, according to an embodiment of the present invention.
[0048] FIG. 5 shows images of 3T3L1 cells taken by inverted light microscopy after the exposure to test compounds. (A) Condition medium, (B) Standard metformin drug (100 µM), and (C) 400 µg/mL of C. forskohlii root extract treated cells for 24h, according to the embodiment of the present invention.
[0049] FIG. 6 shows overlaid expression graph for the presence of fluorescent 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) in given untreated 3T3L1 cells (black color line) and standard drug–treated cells (metformin, 100µM) (red color line) and 100 µg/mL of C. forskohlii root extract treated cells (green color line), according to the embodiments of the present invention.
[0050] FIG. 7 shows overlaid fluorescence intensities of given untreated 3T3L1 cells (Black colour line) and Standard drug treated cells (Metformin 100µM, Red colour line) and 100µg/mL of C. forskohlii (Forcslim™) extract treated cells (Green Colour line), according to an embodiment of the present invention.
[0051] FIG. 8 shows glucose transporter 4 (GLUT4) expression study on 3T3L1 cell line by flow cytometry, according to an embodiment of the present invention.

F) DETAILED DESCRIPTION OF EMBODIMENTS
[0052] In the following detailed description, a reference is made to the accompanying drawings that form a part hereof, and in which the specific embodiments that may be practiced is shown by way of illustration. The embodiments are described in sufficient detail to enable those skilled in the art to practice the embodiments and it is to be understood that the logical, mechanical, electronic and other changes may be made without departing from the scope of the embodiments. The following detailed description is therefore not to be taken in a limiting sense.
[0053] The various embodiments of the presentprovide a composition and method of Coleus forskohlii (Forcslim™) extract free from14-deoxycoleon U compound that causes fatty liver in patients.
[0054] FIG. 1 is a flow chart showing the various steps involved in the method of synthesizing an extract of Coleus forskohlii (Forcslim™), according to an embodiment of the present invention. With respect to FIG. 1, a method of synthesizing an extract of Coleus forskohlii (Forcslim™), comprises taking a raw material(a), carrying out an extraction of the raw material and collecting an extract (b), removing oil from the extract and dissolving the extract in ethanol (c), removing extra oil from the extract (d). The extra oil is removed by carrying out charcoalization of the extract. The extract is filtered (e). The extract is concentrated (f). The extract is crystallizedat 5-10°C for 3 days (g). The extract is filtered (h). The silica gel chromatography of the extract to separate the fraction, wherein the extract obtained is free from 14-deoxycoleon U compound that causes fatty liver in patients (i). The fraction is dried to get C. forskohlii extract (1-99%) (j).
[0055] According to an embodiment of the present invention, the step of performing the silica gel chromatography comprises using a glass column packed with silica column. The length of the silica column is 2 ft and the diameter of the silica column is 2.5 inch. The sample is loaded into the column. The sample is eluted with hexane at flow rate of 1ml/min. The first 10 fractions are discarded and then a gradient elution is started with combination of hexane and diethyl ether from (100:0) to (80:20). The fraction containing 14-Deoxycoleon is collected and eluted with 100% ethyl acetate to get 14-Deoxycoloen free extract. The extract is concentrated to thick paste and added with ethanol. The ethanol is distilled out completely and the residue is dried to get C. forskohlii extract (1-99%).
[0056] According to an embodiment of the present invention, the raw material is prepared by taking the roots of Coleus forskohlii (Forcslim™) plant and grinding the roots to obtain a powder.
[0057] According to an embodiment of the present invention, the extraction is carried out using an extractor and an alcohol, wherein the alcohol has a concentration of 70%.
[0058] According to an embodiment of the present invention, the oil is removed by first washing the extract with water.
[0059] According to an embodiment of the present invention, the extract free from 14-deoxycoleon U is administered to patients for treatment of obesity.
[0060] According to an embodiment of the present invention, the extract is administered in a therapeutically effective amount to a patient for promoting lean body mass in a mammal in need thereof.
[0061] According to an embodiment of the present invention, the extract prevents diet induced liver dysfunction and improves liver function by normalizing the elevated liver enzymes selected from the group consisting of SGOT and SGPT.
[0062] According to an embodiment of the present invention, the extract is free from 14-deoxycoleon U is used for treatment of inflammation in patients.
[0063] According to another embodiment of the present invention, A pharmaceutical composition comprising an extract of Coleus forskohlii (Forcslim™) plant, wherein the composition is effective in treatment of diabetes associated with risk of hyperlipidaemia and cardiovascular disease.
[0064] According to an embodiment of the present invention, a pharmaceutical composition comprising an extract of Coleus forskohlii(Forcslim™) plant, wherein the composition activates enzyme adenylyl cyclase and increases intracellular levels of cAMP, wherein the cAMP stimulates the activity of a protein kinase which phosphorylates a hormone-sensitive lipase to produce the active form of this enzyme.
[0065] According to an embodiment of the present invention, a pharmaceutical composition comprising an extract of Coleus forskohlii(Forcslim™) plant, wherein the composition is formulated into dosage forms selected from a group comprising liquid, powder, granule, capsule, tablet, syrups, elixirs, phytoceuticals and nutraceuticals.
[0066] According to an embodiment of the present invention, a pharmaceutical composition comprising an extract of Coleus forskohlii plant wherein the therapeutically effective amount of the extract is between 0.01 mg and 500 mg per kilogram of body weight of an individual to which the extract is administered.

EXPERIMENTAL DETAILS
In Vitro Anti-Inflammatory ActivityC. Forskohlii (Forcslim™) Root
[0067] In this study, the anti-inflammatory potency of C. forskohlii root extract was evaluated in Lipopolysaccharide (LPS 2µg/ml) stimulated THP1 – Human Peripheral Blood Acute Monocytic leukaemia cell line by measuring the relative fluorescence intensity of cytokines, Interleukin-10(IL-10) by flow cytometric analysis.Cell viability of THP1cells determined by the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay to identify a non-cytotoxic concentration of C. forskohlii(Forcslim™) extract for the respective cell lines after 24 h exposure period. C. forskohlii (Forcslim™) root extract significantly suppressed the anti-inflammatory cytokine expressions of IL-10 in LPS pre-stimulated cells categorizing as a potentially potent anti-inflammatory drug. The mean fluorescence intensity percentage of IL-10 in is control 9.80, LPS 31, 70 and C. forskohlii25, 05 respectively.

[0068] In vitro antidiabetic activity C. forskohlii root(FORCSLIM™)
[0069] This study was aimed at assessment of in vitro antidiabetic activities of ethanolic extract of C. forskohlii root(Forcslim™) by using 3T3L1 cell line. The cytotoxic effect of the root extract was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The glucose uptake–inducing capabilities and its correlation with glucose transporter 4 (GLUT4) translocation were measured by flow cytometry in 3T3L1 cells. In addition, the inhibitory effect of C. forskohlii root extract(Forcslim™) on a-amylase activity was determined by colorimetric methods.Different concentrations of C. forskohlii (Forcslim™) extractdid not show any toxicity on 3T3L1 cells, after the treatment for 24h. On stimulation with root extract, 62.34% and 76.86% of 3T3L1 cells showed glucose uptake and GLUT4 expression, respectively. The colorimetric assays showed that the ethanolic leaf extract of C. forskohlii root has a significant inhibitory effect on the activity of a-amylase enzyme inhibitory concentration (IC50) value of 238.65 µg/mL. On the basis of the results of this study, it is evident that C. forskohlii . forskohlii root extract showed promising anti-diabetic effect when compared to the standard drugs metformin and acarbose and was nontoxic to 3T3L1 cells. Thus, it can be further investigated to recommend as a possible alternative treatment in antidiabetic applications.
In Vivo Hepatoprotective Activity C. Forskohlii Root
[0070] The present study was carried out to assess hepatoprotective potential for ethanolic extract of Coleus forskohlii (Forcslim™) root against paracetamol (PCM 2 g/kg b.w., p.o) induced hepatotoxicity in rats. Oral administration of Coleus forskohlii (Forcslim™) in two doses 250mg/kg and 500mg/kg body weight were subjected for the evaluation of hepatoprotective potential against PCM (2g/kg) induced liver injury.Silymarin (50 mg/kg b.w.) was employed as standard hepatoprotective agent.The biochemical parameters such as serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), glutathione (GSH), super oxide dismutase (SOD), total bilirubin (TBR), total protein (TP) and catalase (CAT) were estimated. In addition, histopathological study was also carried out. The results revealed significant decrease in SGPT, SGOT and TBR and increase in GSH, TP, SOD and CAT levels when compared with toxic control. The ethanolic extract at dose of 500 mg/kg b.w. was found to be more potent than 250 mg/kg. The ethanolic extract of C. forskohlii root(Forcslim™) seems to justify the promising hepatoprotective effect on PCM induced liver damage in rats.
[0071] Hepatoprotective activity of C. forskohlii(Forcslim™)
[0072] The studies were carried out using male wistar albino rats (180-200g). They were obtained from the animal house, Bharathi College of pharmacy Mandya, Karnataka, India. The animals were grouped and housed in polyacrylic cages (38 x 23 x10 cm) with not more than six animals per cage and maintained under standard laboratory conditions (temperature 25 ± 2? C) with dark and light cycle (12/12 h). All the animals were to laboratory condition for a week before commencement of experiment. The ethical clearance was obtained from Institutional Animal Ethics Committee (IAEC) before the experiment (1135/PO/Re/S/07/CPCSEA).
[0073] Study design
[0074] 30 Wistar albino rats of either sex were used for the study. They were randomly divided into 5 groups with six (6) rats in each group.
[0075] Group I: Normal control rats (distilled water given p.o)
[0076] Group II: Distilled water+Paracetamol (2 g/kg bw, orally) (Toxic control)
[0077] Group III: Silymarin (50 mg/kg/day p.o) +Paracetamol (2 g/kg bw, orally) (positive control)
[0078] Group IV:C. forskohlii(Forcslim™) (250 mg/kg/day respectively) +Paracetamol (2 g/kg bw, orally)
[0079] Group V:C. forskohlii(Forcslim™) (500 mg/kg/day respectively) +Paracetamol (2 g/kg bw, orally)
[0080] Biochemical Studies
[0081] The blood samples were drawn from all the animals by puncturing retro-orbital plexus on 10th day of the treatment. Serum was separated by centrifuging blood at 2500 rpm for 15 min and the levels of SGOT, SGPT, Total bilirubin, and total protein were analyzed by using a commercially available enzymatic kit (AGAPPE, India) and an Autoanalyser (Chemistry Analyser (CA 2005),
[0082] Histopathology
[0083] A section of the liver was collected and immediately fixed in 10 % formalin, and then dehydration in ascending grades of alcohol (ethanol) of 70, 80 and 95 % and absolute alcohol for 2 changes each. The tissues were cleared in xylene and embedded in paraffin wax. Serial section of 5-6 microns in thickness were obtained using rotary microtome and stained with hematoxylin and eosin. The stained sections were examined under microscope for analyzing any changes in the architecture of the liver tissue due to paracetamol challenge and improved liver architecture due to pre-treatment with test extract and standard drug.
[0084] Results
[0085] The effect of C. forskohlii (Forcslim™) on various biochemical parameters are shown n Table-1. It was observed that, the activities of serum SGOT, SGPT and TBR were increased GSH, SOD, CAT and TB decreased markedly in paracetamol fed animals as compared to normal control group. The administration of C. forskohlii (Forcslim™) 250mg/kg and 500mg/kg lowered the paracetamol induced elevation of serum parameters. The Standard Silymarin 50mg/kg bw treatment showed extremely significant (P<0.001) reduction in SGPT, SGOT, TBR and increased GSH, SOD and TB. C. forskohlii (Forcslim™) (250mg/kg bw and 500mg/kgbw) treated animals showed moderately significant (P<0.05) reduction in SGPT, SGOT &TBR and increased GSH, SOD and TB levels as compared to toxic control group. Histopathological examination of liver sections of normal rats showed normal hepatic cells with cytoplasm and nucleus whereas paracetamol treated group showed various degrees of fatty degeneration like ballooning of hepatocytes, infiltration of lymphocytes and the loss of cellular boundaries. Administration of C. forskohlii (Forcslim™) at a dose of 500 mg/kg significantly normalized these defects in the histological architecture of the liver.
[0086] Effect of C. forskohlii (Forcslim™) on serum SGPT (AST) level in paracetamol induced hepatotoxicity in albino wistar rats.
[0087] The effect of C. forskohlii (Forcslim™) on SGPT level in serum of paracetamol induced hepatotoxicity in male albino wistar rats. The control had shown the SGPT level in serum of 365±3.4IU/L but after paracetamol treatment, it increased to 816±2.1IU/L. whereas after administration of C. forskohlii(Forcslim™) at the doses of 250 mg/kg, and 500 mg/kg, bw po in paracetamol in toxicated rats, the SGPT level reduced to 721±1IU/L and 536±0.9 IU/L respectively (Table-1).These data suggested that C. forskohlii(Forcslim™) might protect the liver against paracetamol induced injury by attenuating oxidative stress.
[0088] Effect of C. forskohlii (Forcslim™) on serum SGOT level in paracetamol induced hepatotoxicity in albino wistar rats.
[0089] The effect of C. forskohlii (Forcslim™) on SGOT level in serum of paracetamol induced hepatotoxicity in male albino wistar rats. The control had shown the SGOT level in serum of 370±0.56 IU/L but after paracetamol treatment, it increased to 918±2.10 IU/L. Whereas after administration of C. forskohlii(Forcslim™) at the doses of 250 mg/kg and 500 mg/kg, bw po in paracetamol intoxicated rats, the SGOT level reduced to 698± 1.20 IU/L and 558±1.4 IU/L respectively (Table-1).
[0090] Effect of C. forskohlii (Forcslim™) on serum Total bilirubin level in paracetamol induced hepatotoxicity in albino wistar rats.
[0091] The effect of C. forskohlii (Forcslim™) on Total bilirubin level in serum of paracetamol intoxicated male albino rats. The control has showed to bilirubin level in serum of (0.04±0.0003) mg/dL, but after paracetamol, it increased to (0.20±0.008) mg/dL. Whereas after administration of C. forskohlii (Forcslim™) at a dose of 250mg/ kg bw and 500 mg/kg bw in paracetamol intoxicated rats, the bilirubin level reduced to (0.15±0.01) mg/dL and 0.11±0.004 mg/dL. The reduced bilirubin level was also observed 0.04±0.0003 mg/dL control group (Table-1).
[0092] Effects of C. forskohlii (Forcslim™) treatments on liver enzyme activities
[0093] The effect of treatments for antioxidant enzyme activities. Table -1 shows changes of TP, GSH, SOD and CAT activities in the liver tissue indicating liver oxidative damage. Exposure of rats to paracetamol produced significant drops in TP (2.83±0.27), GSH (10±1.1), SOD (20±0.4) and CAT (41±1.1) (P<0.05) enzyme activities compared to other groups respectively. The rats received 250mg/kg.bw C. forskohlii +PCM exhibited significantly elevated levels of TP (3.51±0.07), GSH (17±1.19), SOD (40±1.1) CAT (53.5±0.8) respectively. The administration of 500mg/kg.bw C. forskohlii(Forcslim™) + PCM exhibited significantly increased levels of TP (4.13±0.25), GSH (23±0.95) and SOD (56±1.6) CAT (58.6±0.88) in comparison with a paracetamol group (P < 0.05) but not with the control. In contrast, treatment of C. forskohlii (Forcslim™) resulted in a significant amelioration of the enzyme (TP, GSH, SOD and CAT) activities (Table-1).
[0094] The present study shows that C. forskohlii extract (Forcslim™) possess significant in vivo hepatoprotective activity. Therefore, C. forskohlii extract can be potentially developed into an effective protective agent against acute liver injury.
Table-1 Effect of C. forskohlii (Forcslim™) on biochemical parameters.
Treatment Groups SGOT
(IU/L) SGPT
(IU/L) GSH (/L) TBR(mg/dL) SOD
U/mg protein
CAT TP
Normal (D/W) 370±0.56 365±3.4 30±1.3 0.04±0.0003 68±0.6 67±0.95 5.14±0.24
Silymarin (50 mg/kg) 448±3.16*** 460±2.6*** 26±0.6** 0.10±0.003*** 64±0.6*** 63±0.6* 4.83±.0.19 ns
Paracetamol toxic Control (2g/kg) 918±2.10*** 816±2.1*** 10±1.1*** 0.20±0.008* 20±0.4*** 41±1.1*** 2.83±0.27***
C. forskohlii(Forcslim™) (250 mg/kg) + Paracetamol 698± 1.20*** 721±1.00*** 17±1.19*** 0.15±0.01*** 40±1.1*** 53.5±0.8*** 3.51±0.07***
C. forskohlii(Forcslim™) (500 mg/kg) + Paracetamol 558±1.4*** 536±0.9*** 23±0.95*** 0.11±0.004*** 56±1.6 *** 58.6±0.88*** 4.13±0.25*
[0095] SGOT:serum glutamic-oxaloacetic transaminase, SGPT: serum glutamic pyruvic transaminase, GSH:glutathione, SOD: super oxide dismutase, TBR: total bilirubin, TP: total protein and CAT: catalase
[0096] All values are expressed as mean±S.E.M for 6 rats in each group P ? 0.05 denote value significantly different from control.

In Vivo Anti-Obesity Activity C. Forskohlii Root (Forcslim™)
[0097] Aim of the present study was to evaluate the anti-obesity activity of ethanol extract of C. forskohlii root in mice fed with cafeteria diet. Female Swiss Albino mice were divided into five groups, which received normal and cafeteria diet, standard drug simvastatin (10 mg/kg) and C. forskohlii (250 and 500 mg/kg) daily for 40 days. Parameters such as body weight, body mass index (BMI), Lee index of obesity (LIO), food consumption, serum glucose, triglyceride, total cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), atherogenic index and organ weight were studied for evaluating the anti-obesity activity of C. forskohlii(Forcslim™). As a result there was a significant increase in locomotor activity with HDL and significant decrease in food consumption, body weight, BMI, LIO, total cholesterol, triglyceride, LDL and glucose was seen with C. forskohlii extract (Forcslim™) which is opposite to the result of cafeteria diet. Present findings suggest that the ethanolic extract of C. forskohlii (Forcslim™) have significant anti-obesity activity by maintaining the normal levels of physical and biochemical parameters.
[0098] Material and methods
[0099] Experimental protocol for anti-obesity activity:
[00100] Female Swiss Albino mice (22-26 gm) were randomly divided into six groups of six mice in each and treated as follows:
[00101] Group I: Received standard laboratory feed, i.e., the normal diet.
[00102] Group II: Received cafeteria diet in pellets forms.
[00103] Group III: Simvastatin (10 mg/kg, orally) was administered daily.
[00104] Group IV: C. forskohlii (FORCSLIM™) (250 mg/kg, orally) was administered daily.
[00105] Group V: C. (FORCSLIM™)
[00106] Preparation of cafeteria diet for induction of obesity: The method described by Harris and Kulkarni was followed with some modifications. Cafeteria diet (highly palatable, energy rich animal diet that includes a variety of human snack foods) consists of 3 diets, which includes (condensed milk 48 g + bread 48 g), (chocolate 18 g + biscuits 36 g + dried coconut 36 g), (cheese 48 g + boiled potatoes 60 g). Cafeteria diet was presented in the form of pellets to 5 groups of 6 mice in each for 40 days.
[00107] Parameters evaluated
[00108] Body weight
[00109] Body weights of mice (g) were recorded on 1, 10, 20, 30 and 40 day in each group.
[00110] Body mass index and lee index of obesity: Body mass index (BMI) and lee index of obesity (LIO) of mice were recorded on 1 and 40 day of study (i.e., initial and final body weight and body height) and was measured by using formulas.
BMI = Body weight in gm/(Height in cm)2
LIO = Body weight in gm (1/3)/Nasoanal length in cm
[00111] Food consumption
[00112] Food consumption study was carried out on 1, 10, 20, 30 and 40 day and recorded at 1 h, 2 h and 3 h of time intervals. The food consumption was estimated by substracting the amount of food left on the grid from initial food weight.
[00113] Results
[00114] Effect of C. forskohlii (FORCSLIM™) on body weight: 2. shows the effect of C. forskohlii (FORCSLIM™) on body weight in normal and experimental group of mice. Cafeteria diet group showed significant (P < 0.001) increase in body weight on 10, 20, 30, and 40 day as compared to a normal diet group. Cafeteria diet fed mice treated with simvastatin showed significant (P < 0.05- P < 0.001) decrease in body weight on 10, 20, 30 and 40 day as compared to cafeteria diet group. Oral administration of C. forskohlii (FORCSLIM™) group (500 mg/kg) showed significant (P < 0.05- P < 0.001) reduction in body weight at 20, 30 and 40 day as compared to cafeteria diet group.
Table 2: Effect of C. forskohlii (FORCSLIM™)on body weight in normal and experimental group of mice
Treatment groups Days and body weight (g)
1 day 10 day 20 day 30 day 40 day
Normal diet 22.08±1.24 22.32±1.93 22.91±1.84 23.64±1.98 25.56±2.51
Cafeteria diet 23.87±1.64 26.32±2.93 27.61±2.45 29.43±2.83 31.43±71
Cafeteria diet+simvastatin 23.47±1.56 22.98±2.42 21.03±2.24 21.52±1.67 20.02±1.72
Cafeteria diet+C. forskohlii (FORCSLIM™) 250µg/ml 23.98±1.83 26.86±2.18 25.26±1.04 25.62±1.34 23.21±1.74
Cafeteria diet+C. forskohlii (FORCSLIM™) 500250µg/ml 23.28±1.83 25.47±2.10 24.42±1.77 23.89±1.64 22.56±1.33
[00115] All values are expressed as a mean±SEM, n=6, p?0.001 compared with normal diet group and p?0.005, p?0.001 compared with compared with cafeteria diet group
[00116] Effect of C. forskohlii (FORCSLIM™) on BMI (Body mass index) and LIO(Lee index of obesity): Effect of C. forskohlii on BMI and LIO in normal and experimental group of mice is shown in Table 3. Feeding the cafeteria diet to mice was found to significantly (P < 0.001) increase the final BMI and LIO when compared to mice fed with normal diet. Cafeteria diet fed mice treated with simvastatin, C. forskohlii (Forcslim™) (500 mg/kg) groups showed a significant (P < 0.001) decrease in the final BMI and LIO when compared with cafeteria diet group.
Table 3. Effect of C. forskohlii (FORCSLIM™) ON BMI and LIO in normal and experimental group of mice

Treatment groups Initial BMI g/cm2 Final BMI g/cm2 Initial LIO g/cm2 Final LIOg/cm2
Normal diet 0.40±0.02 0.44±0.03 60.1±3.1 65.6±5.2
Cafeteria diet 0.42±0.03 0.56±0.06 63.8±0.02 81.3±3.6
Cafeteria diet+simvastatin 0.41±0.03 0.35±0.02 62.93±4.3 51.8±3.6
Cafeteria diet+C. forskohlii 250mg/kg 0.42±0.03 0.42±0.03 61.8±4.8 64.2±4.6
Cafeteria diet+C. forskohlii 500mg/kg 0.42±0.03 0.37±0.02 63.2±4 56.9±3.6
[00117] All values are expressed as a mean±SEM, n=6, BMI: Body mass index, LIO: Lee index of obesity, p?0.001 compared with normal diet group and p?0.005, p?0.001 compared with compared with cafeteria diet group
[00118] Effect of C. forskohlii (FORCSLIM™)on food consumption: Table 4 shows the effect of C. forskohlii(Forcslim™) on food consumption in normal and experimental group of mice. Food consumption was found to be significantly (P < 0.001) increased on 1, 10, 20, 30 and 40 day as compared to a normal diet group. Simvastatin administered mice showed a significant (P < 0.01, P < 0.001) decrease in food consumption on 30 and 40 day as compared to cafeteria diet group. The C. forskohlii(Forcslim™) (500 mg/kg) treated mice showed significantly (P < 0.05, P < 0.01) decreased food consumption on 30 and 40 day as compared to cafeteria diet group.
Table 4: Effect of C. forskohlii (FORCSLIM™)on food consumption in normal and experimental group of mice

Treatment groups Days and foodconsumption (g)
1 day 10 day 20 day 30 day 40 day
Normal diet 2.67±0.16 1.86±0.20 3.27±0.2 2.10±0.17 3.39±0.31
Cafeteria diet 15.9±1.74 14.5±1.42 13.1±1.23 11.92±1.03 10.2±.90
Cafeteria diet+simvastatin 16.9±1.56 12.7±1.28 10.5±.98 8.90±.80 6.2±0.43
Cafeteria diet+C. forskohlii 17.2±1.73 13.9±1.84 13.8±1.29 10.6±1.04 8.93±0.85
Cafeteria diet+C. forskohlii 16.8±1.83 12.6±1.34 10.45±0.76 9.94±0.89 7.9±0.93
[00119] All values are expressed as a mean±SEM, n=6, p?0.001 compared with normal diet group and p?0.005, p?0.001 compared with compared with cafeteria diet group
[00120] Effect of C. forskohlii (FORCSLIM™) on biochemical profile: Effect of C. forskohlii (Forcslim™)on biochemical profile in normal and experimental group of mice is shown in Table 5. Feeding the cafeteria diet to mice was found to significantly (P < 0.001) increase the levels of serum glucose, triglycerides, total cholesterol, LDL, atherogenic index and significantly (P < 0.001) decrease the level of HDL when compared to mice fed with normal diet. Cafeteria diet fed mice treated with simvastatin, C. forskohlii (500 mg/kg) groups showed a significant (P < 0.001) decrease in the levels of serum glucose, triglycerides, total cholesterol, LDL, AI and significant (P < 0.001) increase in the levels of HDL when compared with cafeteria diet group.
Table 5. Effect of C. forskohlii (FORCSLIM™) on biochemical profile in normal and experimental group of mice
Treatment groups Glucose
(mg/ml) Triglycerides
(mg/ml) Total cholesterol
(mg/ml) HDL
(mg/ml) LDL
(mg/ml) AI
(Units)
Normal diet 75.2±2.5 65.73±7.2 165.8±10.3 38.2±3.5 110.8±11.6 3.42±0.72
Cafeteria diet 95.3±7.9 98.87±8.9 195.6±14.9 29.3±22.8 156±0.6 7.07±0.8
Cafeteria diet+simvastatin 78.4±9.6 71.2±4.7 173.8±13.8 36.2±2.1 124.2±10.2 3.98±0.5
Cafeteria diet+C. forskohlii 250mg/kg 81.2±6.9 84.9±9.2 189.2±12.7 30.1±2.7 143.2±11,6 5.27±0.4
Cafeteria diet+C. forskohlii 500mg/kg 75.9±8.5 76.9±6.3 178.2±12.8 35.2±2.4 129.4±11,3 4.29±0.7
[00121] All values are expressed as a mean±SEM, n=6, HDL: High density lipoprotein, LDL: Low density lipoprotein, AI: Atherogenic index, p?0.001 compared with normal diet group and p?0.005, p?0.001 compared with cafeteria diet group.
[00122] Effect of C. forskohlii (FORCSLIM™)on organ weight:Table 6 shows the effect of C. forskohlii (Forcslim™)on organ weight in normal and experimental group of mice. The weight of organs such as liver and small intestine in cafeteria diet group was found to be significantly (P < 0.001) increased as compared to a normal diet group. Simvastatin administered mice showed a significant decrease in organ weight such as liver (P < 0.01) and small intestine (P < 0.001) as compared to cafeteria diet group. The C. forskohlii (Forcslim™) (500 mg/kg) treated mice showed significantly (P < 0.05) decreased weight of liver and small intestine as compared to cafeteria diet group. There was no significant difference between the organ weights of brain, stomach, heart, lungs and kidneys of cafeteria diet group and other experimental groups.
Table 6: Effect of C. forskohlii (FORCSLIM™)on organ weight in normal and experimental group of mice

Treatment groups Brain(g) Liver(g) Stomach(g) Heart(g) Lungs(g) Kidney
Right Left Right Left
Normal diet 0.29±0.03 0.98±0.04 0.28±0.03 0.08±0.004 0.16±0.002 0.16±0.005 0.12±0.004 0.12±0.004
Cafeteria diet 0.30±0.03 1.17±0.05 0.50±0.05 0.17±0.006 0.15±0.002 0.15±0.005 0.14±0.003 0.14±0.003
Cafeteria diet+simvastatin 0.29±0.03 1.7±0.04 0.32±0.04 0.12±0.003 0.18±0.002 0.18±0.006 0.11±0.003 0.11±0.003
Cafeteria diet+C. forskohlii
250mg/kg 0.28±0.03 1.18±0.04
0.37±0.03 0.14±0.004 0.17±0.002 0.17±0.004 0.11±0.003 0.11±0.003
Cafeteria diet+C. forskohlii 500mg/kg 0.28±0.03 1.14±0.04
0.38±0.03 0.13±0.08 0.18±0.002 0.18±0.002 0.12±0.003 0.12±0.003
All values are expressed as a mean±SEM, n=6, p?0.001 compared with normal diet group and p?0.005, p?0.001 compared with cafeteria diet group.

ANTI-OBESITY ACTIVITY OF ETHANOLIC EXTRACT OF C. FORSKOHLII ROOT IN MICE FED WITH CAFETERIA DIET
[00123] Aim of the present study was to evaluate the anti-obesity activity of ethanol extract of C. forskohlii root in mice fed with cafeteria diet. Female Swiss Albino mice were divided into five groups, which received normal and cafeteria diet, standard drug simvastatin (10 mg/kg) and C. forskohlii (Forcslim™) (250 and 500 mg/kg) daily for 40 days. Parameters such as body weight, body mass index (BMI), Lee index of obesity (LIO), food consumption, serum glucose, triglyceride, total cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), atherogenic index and organ weight were studied for evaluating the anti-obesity activity of C. forskohlii.As a result there was a Significant increase in locomotor activity with HDL and significant decrease in food consumption, body weight, BMI, LIO, total cholesterol, triglyceride, LDL and glucose was seen with C. forskohlii extract(Forcslim™) which is opposite to the result of cafeteria diet. Present findings suggest that the ethanolic extract of C. forskohlii (Forcslim™)have significant anti-obesity activity by maintaining the normal levels of physical and biochemical parameters.
[00124] Material and methods:
[00125] Experimental protocol for anti-obesity activity:
[00126] Female Swiss Albino mice (22-26 gm) were randomly divided into six groups of six mice in each and treated as follows:
[00127] Group I: Received standard laboratory feed, i.e., the normal diet.
[00128] Group II: Received cafeteria diet in pellets forms.
[00129] Group III: Simvastatin (10 mg/kg, orally) was administered daily.
[00130] Group IV: C. forskohlii (250 mg/kg, orally) was administered daily.
[00131] Group V: C. forskohlii (FORCSLIM™)
[00132] Preparation of cafeteria diet for induction of obesity:The method described by Harris and Kulkarni was followed with some modifications. Cafeteria diet (highly palatable, energy rich animal diet that includes a variety of human snack foods) consists of 3 diets, which includes (condensed milk 48 g + bread 48 g), (chocolate 18 g + biscuits 36 g + dried coconut 36 g), (cheese 48 g + boiled potatoes 60 g). Cafeteria diet was presented in the form of pellets to 5 groups of 6 mice in each for 40 days.
[00133] Parameters evaluated
[00134] Body weight: Body weights of mice (g) were recorded on 1, 10, 20, 30 and 40 day in each group.
[00135] Body mass index and lee index of obesity: Body mass index (BMI) and lee index of obesity (LIO) of mice were recorded on 1 and 40 day of study (i.e., initial and final body weight and body height) and was measured by using formulas.
BMI = Body weight in gm/(Height in cm)2
LIO = Body weight in gm (1/3)/Nasoanal length in cm
[00136] Food consumption:Food consumption study was carried out on 1, 10, 20, 30 and 40 day and recorded at 1 h, 2 h and 3 h of time intervals. The food consumption was estimated by substracting the amount of food left on the grid from initial food weight.
[00137] Results
[00138] Effect of C. forskohliion body weight: Table 7. shows the effect of C. forskohliion body weight in normal and experimental group of mice. Cafeteria diet group showed significant (P < 0.001) increase in body weight on 10, 20, 30, and 40 day as compared to a normal diet group. Cafeteria diet fed mice treated with simvastatin showed significant (P < 0.05- P < 0.001) decrease in body weight on 10, 20, 30 and 40 day as compared to cafeteria diet group. Oral administration of C. forskohlii (FORCSLIM™) group (500 mg/kg) showed significant (P < 0.05- P < 0.001) reduction in body weight at 20, 30 and 40 day as compared to cafeteria diet group.
Table 7: Effect of C. forskohlii (FORCSLIM™) ONbody weight in normal and experimental group of mice
Treatment groups Days and body weight (g)
1 day 10 day 20 day 30 day 40 day
Normal diet 22.08±1.24 22.32±1.93 22.91±1.84 23.64±1.98 25.56±2.51
Cafeteria diet 23.87±1.64 26.32±2.93 27.61±2.45 29.43±2.83 31.43±71
Cafeteria diet+simvastatin 23.47±1.56 22.98±2.42 21.03±2.24 21.52±1.67 20.02±1.72
Cafeteria diet+C. forskohlii (FORCSLIM™) 250µg/ml 23.98±1.83 26.86±2.18 25.26±1.04 25.62±1.34 23.21±1.74
Cafeteria diet+C. forskohlii (FORCSLIM™) 500250µg/ml 23.28±1.83 25.47±2.10 24.42±1.77 23.89±1.64 22.56±1.33
[00139] All values are expressed as a mean±SEM, n=6, p?0.001 compared with normal diet group and p?0.005, p?0.001 compared with compared with cafeteria diet group
[00140] Effect of C. forskohlii (FORCSLIM™)onBMI(Body mass index) and LIO(Lee index of obesity):Effect of C. forskohlii on BMI and LIO in normal and experimental group of mice is shown in Table 8. Feeding the cafeteria diet to mice was found to significantly (P < 0.001) increase the final BMI and LIO when compared to mice fed with normal diet. Cafeteria diet fed mice treated with simvastatin, C. forskohlii (500 mg/kg) groups showed a significant (P < 0.001) decrease in the final BMI and LIO when compared with cafeteria diet group.
Table 8: Effect of C. forskohlii (FORCSLIM™)on BMI and LIO in normal and experimental group of mice
Treatment groups Initial BMI g/cm2 Final BMI g/cm2 Initial LIO g/cm2 Final LIOg/cm2
Normal diet 0.40±0.02 0.44±0.03 60.1±3.1 65.6±5.2
Cafeteria diet 0.42±0.03 0.56±0.06 63.8±0.02 81.3±3.6
Cafeteria diet+simvastatin 0.41±0.03 0.35±0.02 62.93±4.3 51.8±3.6
Cafeteria diet+C. forskohlii 250mg/kg 0.42±0.03 0.42±0.03 61.8±4.8 64.2±4.6
Cafeteria diet+C. forskohlii 500mg/kg 0.42±0.03 0.37±0.02 63.2±4 56.9±3.6
[00141] All values are expressed as a mean±SEM, n=6, BMI: Body mass index, LIO: Lee index of obesity, p?0.001 compared with normal diet group and p?0.005, p?0.001 compared with compared with cafeteria diet group
[00142] Effect of C. forskohlii(FORCSLIM™)on food consumption: Table 9 shows the effect of C. forskohlii on food consumption in normal and experimental group of mice. Food consumption was found to be significantly (P < 0.001) increased on 1, 10, 20, 30 and 40 day as compared to a normal diet group. Simvastatin administered mice showed a significant (P < 0.01, P < 0.001) decrease in food consumption on 30 and 40 day as compared to cafeteria diet group. The C. forskohlii (500 mg/kg) treated mice showed significantly (P < 0.05, P < 0.01) decreased food consumption on 30 and 40 day as compared to cafeteria diet group.
Table 9: Effect of C. forskohlii (FORCSLIM™) on food consumption in normal and experimental group of mice
Treatment groups Days and food consumption (g)
1 day 10 day 20 day 30 day 40 day
Normal diet 2.67±0.16 1.86±0.20 3.27±0.2 2.10±0.17 3.39±0.31
Cafeteria diet 15.9±1.74 14.5±1.42 13.1±1.23 11.92±1.03 10.2±.90
Cafeteria diet+simvastatin 16.9±1.56 12.7±1.28 10.5±.98 8.90±.80 6.2±0.43
Cafeteria diet+C. forskohlii 17.2±1.73 13.9±1.84 13.8±1.29 10.6±1.04 8.93±0.85
Cafeteria diet+C. forskohlii 16.8±1.83 12.6±1.34 10.45±0.76 9.94±0.89 7.9±0.93
[00143] All values are expressed as a mean±SEM, n=6, p?0.001 compared with normal diet group and p?0.005, p?0.001 compared with compared with cafeteria diet group.
[00144] Effect of C. forskohlii (FORCSLIM™) on biochemical profile:Effect of C. forskohlii on biochemical profile in normal and experimental group of mice is shown in Table 10. Feeding the cafeteria diet to mice was found to significantly (P < 0.001) increase the levels of serum glucose, triglycerides, total cholesterol, LDL, atherogenic index and significantly (P < 0.001) decrease the level of HDL when compared to mice fed with normal diet. Cafeteria diet fed mice treated with simvastatin, C. forskohlii (500 mg/kg) groups showed a significant (P < 0.001) decrease in the levels of serum glucose, triglycerides, total cholesterol, LDL, AI and significant (P < 0.001) increase in the levels of HDL when compared with cafeteria diet group.
Table 10: Effect of C. forskohlii (FORCSLIM™)on biochemical profile in normal and experimental group of mice
Treatment groups Glucose
(mg/ml) Triglycerides
(mg/ml) Total cholesterol
(mg/ml) HDL
(mg/ml) LDL
(mg/ml) AI
(Units)
Normal diet 75.2±2.5 65.73±7.2 165.8±10.3 38.2±3.5 110.8±11.6 3.42±0.72
Cafeteria diet 95.3±7.9 98.87±8.9 195.6±14.9 29.3±22.8 156±0.6 7.07±0.8
Cafeteria diet+simvastatin 78.4±9.6 71.2±4.7 173.8±13.8 36.2±2.1 124.2±10.2 3.98±0.5
Cafeteria diet+C. forskohlii 250mg/kg 81.2±6.9 84.9±9.2 189.2±12.7 30.1±2.7 143.2±11,6 5.27±0.4
Cafeteria diet+C. forskohlii 500mg/kg 75.9±8.5 76.9±6.3 178.2±12.8 35.2±2.4 129.4±11,3 4.29±0.7
[00145] All values are expressed as a mean±SEM, n=6, HDL: High density lipoprotein, LDL: Low density lipoprotein, AI: Atherogenic index, p?0.001 compared with normal diet group and p?0.005, p?0.001 compared with cafeteria diet group.
[00146] Effect of C. forskohlii (FORCSLIM™)on organ weight
[00147] Table 11 shows the effect of C. forskohlii on organ weight in normal and experimental group of mice. The weight of organs such as liver and small intestine in cafeteria diet group was found to be significantly (P < 0.001) increased as compared to a normal diet group. Simvastatin administered mice showed a significant decrease in organ weight such as liver (P < 0.01) and small intestine (P < 0.001) as compared to cafeteria diet group. The C. forskohlii (500 mg/kg) treated mice showed significantly (P < 0.05) decreased weight of liver and small intestine as compared to cafeteria diet group. There was no significant difference between the organ weights of brain, stomach, heart, lungsand kidneysof cafeteria diet group and other experimental groups.
Table 11: Effect of C. forskohlii (FORCSLIM™) on organ weight in normal and experimental group of mice
Treatment groups Brain(g) Liver(g) Stomach(g) Heart(g) Lungs(g) Kidney
Right Left Right Left
Normal diet 0.29±0.03 0.98±0.04 0.28±0.03 0.08±0.004 0.16±0.002 0.16±0.005 0.12±0.004 0.12±0.004
Cafeteria diet 0.30±0.03 1.17±0.05 0.50±0.05 0.17±0.006 0.15±0.002 0.15±0.005 0.14±0.003 0.14±0.003
Cafeteria diet+simvastatin 0.29±0.03 1.7±0.04 0.32±0.04 0.12±0.003 0.18±0.002 0.18±0.006 0.11±0.003 0.11±0.003
Cafeteria diet+C. forskohlii
250mg/kg 0.28±0.03 1.18±0.04
0.37±0.03 0.14±0.004 0.17±0.002 0.17±0.004 0.11±0.003 0.11±0.003
Cafeteria diet+C. forskohlii 500mg/kg 0.28±0.03 1.14±0.04
0.38±0.03 0.13±0.08 0.18±0.002 0.18±0.002 0.12±0.003 0.12±0.003
[00148] All values are expressed as a mean±SEM, n=6, p?0.001 compared with normal diet group and p?0.005, p?0.001 compared with cafeteria diet group.
Evaluation of Hepatoprotective Activity of COLEUS FORSKOHLII (Forcslim™)ON Paracetamol-Induced Liver Toxicity In Rats
[00149] The present study was carried out to assess hepatoprotective potential for ethanolic extract of Coleus forskohlii root (Forcslim™) against paracetamol (PCM 2g/kg b.w., p.o) induced hepatotoxicity in rats. Oral administration of Coleus forskohlii (Forcslim™) in two doses 250mg/kg and 500mg/kg body weight were subjected for the evaluation of hepatoprotective potential against PCM (2g/kg) induced liver injury. Silymarin (50 mg/kg b.w.) was employed as standard hepatoprotective agent. The biochemical parameters such as serum glutamic-oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), glutathione (GSH), super oxide dismutase (SOD), total bilirubin (TBR), total protein (TP) and catalase (CAT) were estimated. In addition, histopathological study was also carried out. The results revealed significant decrease in SGPT, SGOT and TBR and increase in GSH, TP, SOD and CAT levels when compared with toxic control. The ethanolic extract at dose of 500 mg/kg b.w. was found to be more potent than 250 mg/kg. The ethanolic extract of Forcslim™ seems to justify the promising hepatoprotective effect on PCM induced liver damage in rats.
[00150] Hepatoprotective activity of C. forskohlii(Forcslim™): The studies were carried out using male wistar albino rats (180-200g). They were obtained from the animal house, Bharathi College of pharmacy Mandya, Karnataka, India. The animals were grouped and housed in polyacrylic cages (38 x 23 x10 cm) with not more than six animals per cage and maintained under standard laboratory conditions (temperature 25 ± 2? C) with dark and light cycle (12/12 h). All the animals were to laboratory condition for a week before commencement of experiment. The ethical clearance was obtained from Institutional Animal Ethics Committee (IAEC) before the experiment (1135/PO/Re/S/07/CPCSEA).
[00151] Study design
[00152] 30 Wistar albino rats of either sex were used for the study. They were randomly divided into 5 groups with six (6) rats in each group.
[00153] Group I: Normal control rats (distilled water given p.o)
[00154] Group II: Distilled water+Paracetamol (2 g/kg bw, orally) (Toxic control)
[00155] Group III: Silymarin (50 mg/kg/day p.o) +Paracetamol (2 g/kg bw, orally) (positive control)
[00156] Group IV: C. forskohlii (Forcslim™) (250 mg/kg/day respectively) +Paracetamol (2 g/kg bw, orally)
[00157] Group V: C. forskohlii (Forcslim™) (500 mg/kg/day respectively) +Paracetamol (2 g/kg bw, orally)
[00158] Biochemical Studies:The blood samples were drawn from all the animals by puncturing retro-orbital plexus on 10th day of the treatment. Serum was separated by centrifuging blood at 2500 rpm for 15 min and the levels of SGOT, SGPT, Total bilirubin, and total protein were analyzed by using a commercially available enzymatic kit (AGAPPE, India) and an Autoanalyser (Chemistry Analyser (CA 2005),
[00159] Histopathology:A section of the liver was collected and immediately fixed in 10 % formalin, and then dehydration in ascending grades of alcohol (ethanol) of 70, 80 and 95 % and absolute alcohol for 2 changes each. The tissues were cleared in xylene and embedded in paraffin wax. Serial section of 5-6 microns in thickness were obtained using rotary microtome and stained with hematoxylin and eosin. The stained sections were examined under microscope for analyzing any changes in the architecture of the liver tissue due to paracetamol challenge and improved liver architecture due to pre-treatment with test extract and standard drug.

Table-12 Effect of C. forskohlii(Forcslim™) on biochemical parameters.
Treatment Groups SGOT
(IU/L) SGPT
(IU/L) GSH (/L) TBR(mg/dL) SOD
U/mg protein
CAT TP
Normal (D/W) 370±0.56 365±3.4 30±1.3 0.04±0.0003 68±0.6 67±0.95 5.14±0.24
Silymarin (50 mg/kg) 448±3.16*** 460±2.6*** 26±0.6** 0.10±0.003*** 64±0.6*** 63±0.6* 4.83±.0.19 ns
Paracetamol toxic Control (2g/kg) 918±2.10*** 816±2.1*** 10±1.1*** 0.20±0.008* 20±0.4*** 41±1.1*** 2.83±0.27***
C. forskohlii(Forcslim™) (250 mg/kg) + Paracetamol 698± 1.20*** 721±1.00*** 17±1.19*** 0.15±0.01*** 40±1.1*** 53.5±0.8*** 3.51±0.07***
C. forskohlii(Forcslim™) (500 mg/kg) + Paracetamol 558±1.4*** 536±0.9*** 23±0.95*** 0.11±0.004*** 56±1.6 *** 58.6±0.88*** 4.13±0.25*
[00160] SGOT:serum glutamic-oxaloacetic transaminase, SGPT: serum glutamic pyruvic transaminase, GSH:glutathione, SOD: super oxide dismutase, TBR: total bilirubin, TP: total protein and CAT: catalase
[00161] All values are expressed as mean±S.E.M for 6 rats in each group P ? 0.05 denote value significantly different from control.
[00162] Results: The effect of C. forskohlii(Forcslim™) on various biochemical parameters are shown in Table-12. It was observed that, the activities of serum SGOT, SGPT and TBR were increased GSH, SOD, CAT and TB decreased markedly in paracetamol fed animals as compared to normal control group. The administration of C. forskohlii(Forcslim™) 250mg/kg and 500mg/kg lowered the paracetamol induced elevation of serum parameters. The Standard Silymarin 50mg/kg bw treatment showed extremely significant (P<0.001) reduction in SGPT, SGOT, TBR and increased GSH, SOD and TB. C. forskohlii (Forcslim™) (250mg/kg bw and 500mg/kgbw) treated animals showed moderately significant (P<0.05) reduction in SGPT, SGOT &TBR and increased GSH, SOD and TB levels as compared to toxic control group. Histopathological examination of liver sections of normal rats showed normal hepatic cells with cytoplasm and nucleus whereas paracetamol treated group showed various degrees of fatty degeneration like ballooning of hepatocytes, infiltration of lymphocytes and the loss of cellular boundaries. Administration of C. forskohlii(Forcslim™) at a dose of 500 mg/kg significantly normalized these defects in the histological architecture of the liver.
[00163] Effect of C. forskohlii(Forcslim™) on serum SGPT (AST) level in paracetamol induced hepatotoxicity in albino wistar rats: The effect of C. forskohlii(Forcslim™) on SGPT level in serum of paracetamol induced hepatotoxicity in male albino wistar rats. The control had shown the SGPT level in serum of 365±3.4IU/L but after paracetamol treatment, it increased to 816±2.1IU/L. whereas after administration of C. forskohlii(Forcslim™) at the doses of 250 mg/kg, and 500 mg/kg, bw po in paracetamol in toxicated rats, the SGPT level reduced to 721±1IU/L and 536±0.9 IU/L respectively(Table-12).These data suggested that C. forskohlii(Forcslim™) might protect the liver against paracetamol induced injury by attenuating oxidative stress.
[00164] Effect of C. forskohlii(Forcslim™)on serum SGOT level in paracetamol induced hepatotoxicity in albino wistar rats:The effect of C. forskohlii(Forcslim™) on SGOT level in serum of paracetamol induced hepatotoxicity in male albino wistar rats. The control had shown the SGOT level in serum of 370±0.56 IU/L but after paracetamol treatment, it increased to 918±2.10 IU/L. Whereas after administration of C. forskohlii(Forcslim™) at the doses of 250 mg/kg and 500 mg/kg, bw po in paracetamol intoxicated rats, the SGOT level reduced to 698± 1.20 IU/L and 558±1.4 IU/L respectively (Table-12).
[00165] Effect of C. forskohlii(Forcslim™) on serum Total bilirubin level in paracetamol induced hepatotoxicity in albino wistar rats: The effect of C. forskohlii on Total bilirubin level in serum of paracetamol intoxicated male albino rats. The control has showed to bilirubin level in serum of (0.04±0.0003) mg/dL, but after paracetamol, it increased to (0.20±0.008) mg/dL. Whereas after administration of C. forskohlii(Forcslim™) at a dose of 250mg/ kg bw and 500 mg/kg bw in paracetamol intoxicated rats, the bilirubin level reduced to (0.15±0.01) mg/dL and 0.11±0.004 mg/dL. The reduced bilirubin level was also observed 0.04±0.0003 mg/dL control group(Table-12).
[00166] Effects of C. forskohlii (Forcslim™) treatments on liver enzyme activities: The effect of treatments for antioxidant enzyme activities. Table 12 shows changes of TP, GSH, SOD and CAT activities in the liver tissue indicating liver oxidative damage. Exposure of rats to paracetamol produced significant drops in TP (2.83±0.27), GSH (10±1.1), SOD (20±0.4) and CAT (41±1.1) (P<0.05) enzyme activities compared to other groups respectively. The rats received 250mg/kg.bw C. forskohlii +PCM exhibited significantly elevated levels of TP (3.51±0.07), GSH (17±1.19), SOD (40±1.1) CAT (53.5±0.8) respectively. The administration of 500mg/kg.bw C. forskohlii(Forcslim™) + PCM exhibited significantly increased levels of TP (4.13±0.25), GSH (23±0.95) and SOD (56±1.6) CAT (58.6±0.88) in comparison with a paracetamol group (P < 0.05) but not with the control. In contrast, treatment of C. forskohlii(Forcslim™) resulted in a significant amelioration of the enzyme (TP, GSH, SOD and CAT) activities (Table-12).
[00167] The present study shows that C. forskohlii extract (Forcslim™) possess significant in vivo hepatoprotective activity. Therefore, C. forskohlii extract can be potentially developed into an effective protective agent against acute liver injury.
Anti-Inflammatory Activity OfEthanolic Extract Of Coleus ForskohliiRoot (Forcslim™)

[00168] In this study, the anti-inflammatory potency of FORCSLIM™ was evaluated in Lipopolysaccharide (LPS 2µg/ml) stimulated THP1 – Human Peripheral Blood Acute Monocytic leukemia cell line by measuring the relative fluorescence intensity of cytokines, Interleukin-10(IL-10) by flow cytometric analysis. Cell viability of THP1cells determined by the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay to identify a non-cytotoxic concentration of FORCSLIM™ for the respective cell lines after 24 h exposure period. FORCSLIM™ significantly suppressed the anti-inflammatory cytokine expressions of IL-10 in LPS pre-stimulated cells categorizing as a potentially potent anti-inflammatory drug. The mean fluorescence intensity percentage of IL-10 in is control 9.80, LPS 31, 70 and C. forkohlii25, 05 respectively.
[00169] MTT assay: An MTT assay was performed to evaluate the THP1 cell viability. 20,000 cells per well were seeded in 96 well plate and treated with 6.25, 12.5, 25, 50 and 100 µg/ml of C. forkohlii for 24 h to determine the optimum concentration for further studies. Post incubation period, remove the spent media and add 100 µl of 0.5 mg/ml MTT reagent for 4 h at 37 °C, the formazan crystals generated due to the reduction of MTT by metabolically active cells. They were released from the cells and dissolved using 100 µl DMSO (Sigma-Aldrich, USA). In MTT assay, formazan accumulation directly reflects the mitochondrial activity in the live cells, which is an indirect measurement for the cell viability. The plate was agitated on a gyratory shaker for 10-20 min and the absorbance was measured at 570 nm with an ELISA microplate reader (Biotek, USA). The percentage of cell viability is calculated using the below formula:
% of viable cells = Absorbance sample - Absorbance Blank x 100
Absorbance control - Absorbance Blank
[00170] Anti-inflammatory activity: For the evaluation of anti-inflammatory activity, THP 1 – Human Peripheral Blood Acute Monocytic leukemia cell line were cultured in a 6 well plate and treated with 3 different culture conditions viz., LPS(2µg /ml), LPS (2µg/ml)+FORCSLIM™ (50µg/ml) and untreated without any treatment. Briefly, the cells were pre stimulated with 2 µg/ml of LPS for 3 h to induce inflammation and following stimulation, the cells were either treated with 50µg/ml of FORCSLIM™ or LPS stimulated alone (negative control) with 2 ml DMEM medium. Cells were incubated for 24 h and harvested into centrifuge tubes (BD Biosciences) and centrifuged at 300 × g for five minutes in a Remi: R-8 °C centrifuge and were washed twice with DPBS. The pelleted cells were incubated at room temperature with 0.5 ml BD Cytofix/Cytoperm for 10 min and washed with 0.5% Bovine Serum Albumin solution (1x PBS and 0.1% sodium azide). Cells were incubated with 20µl of PE-Mouse Anti Human interleukin 10(IL-10) separately for 30 min in the dark at 25 °C and expression measured using a BD FACS Calibur flow cytometer (BD Biosciences) and data analyzed by Cell Quest Pro software version 6.
[00171] Statistical analysis: All the data were analyzed using Microsoft Excel 2007 version in creating graphical representation of the mean with calculated standard errors. Flow Cytometric data was analyzed using Cell Quest Pro software version 6.
[00172] Results: Anti-inflammatory activity:
[00173] FORCSLIM™ exhibits significant anti-inflammatory effect through IL-10 inhibition in LPS stimulated macrophage cells. LPS induced inflammation in in vitro cell lines represents a standard paradigm for studying inflammation. The current study shows, the ability of FORCSLIM™ to elicit anti-inflammatory effects on THP-1 cells by evaluating anti-inflammatory cytokines, IL-10 expression. LPS stimulated cells alone, exhibited 2-3 times higher expression than the untreated group. However, the FORCSLIM™treated cells following LPS stimulation were expressing lower expressions than the LPS alone treated cells. The mean fluorescence intensity percentage of IL-10 control 9.80, LPS 11.43, FORCSLIM™ 25.05 and standard 31.70. This study’s results significantly indicated that C. forkohlii root extract (Forcslim™)is a potent and promising natural compound for the treatment of inflammatory related diseases.
[00174] FIG. 2 is a flow cytometry histogram of mean anti-inflammatory cytokines (IL-10) in THP1 cells pre-stimulated with LPS followed by 24 h exposure to FORCSLIM™, according to an embodiment of the present invention. LPS served as a positive control (n=3, mean±standard error).
In Vitro Hepatoprotective Effect OfColeus Forskohlii (Forcslim™) on Ethanol-Induced Oxidative Damage In Hepg2 Cells
[00175] The current study was aimed to evaluate the ethanolic extract of Coleus forskohlii(FORCSLIM™) for its in vitro hepatoprotective activity against ethanol in HepG2 cell lines. In this regard, the cytotoxicity studies were conducted for the extract, C. forskohlii(FORCSLIM™) using 3-(4,5-dimethythiazol- 2-yl)-2,5-diphenyl tetrazolium bromide assay to determine the inhibitory concentration 50% value based on which, the doses 25, 50, 100, 200 and 400µg/ml were selected for the hepatoprotective studies in HepG2 cell lines. The toxicity was induced using ethanol (100mM). The in vitro hepatoprotective activity of the extract was assessed based on the changes in the level of biochemical parameters such as aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase. The C. forskohlii(FORCSLIM™)has shown a dose-dependent cytoprotective activity with maximum protection at 400 µg/ml. The percentage cell viability of the extract, C. forskohlii (FORCSLIM™) at 400 µg/ml was more, i.e., 70.32% which was well comparable to that of standard drug, silymarin (50 µg/ml).
[00176] Methods
[00177] Chemicals
[00178] HepG2 Cell lines were obtained from National Centre for Cell Sciences, Pune India. The drugs and chemicals were purchased from various companies and the details are as follows: Dulbecco’s modified eagles medium, silymarin - Sigma Aldrich, Spruce Street, St. Louis, China; FC reagent, Biochemical kits - Merck Specialties Private Limited, Mumbai, India; fetal bovine serum was purchased from Himedia laboratories, Mumbai, India; Ethanol - Changshu Yangyuan Chemicals, China. All other chemicals and solvents used were of analytical grade.
[00179] Determination of in vitro cytotoxic activity: The 50% cytotoxic concentration (CTC 50) was determined by estimating mitochondrial synthesis using tetrazolium assay. HepG2 cells (5.0 × 103 cells/well) were maintained in 96 well culture plate for 72 hrs in presence of 100 µl of C. forskohlii at the concentrations of 25, 50, 100, 300, 1000, and 2000 µg/ml. At the end of incubation period, the drug solutions in the wells were discarded and 50 µl of 3-(4,5-dimethythiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) prepared in modified Eagle’s medium (MEM) without phenol red was added in each well. The plates were gently shaken and incubated for 3 hrs at 37°C in 5% CO2 atmosphere. After 3 hrs, the supernatant was removed. Later on, 50 µl of propanol was added, and the plates were gently shaken to solubilize the formed formazan followed by 30 min incubation at room temperature with constant shaking. Absorbance (optical density [OD]) was read at 540 nm using microplate reader (Bio-Tek Instruments, Inc., Winooski, VT). The percentage growth inhibition was calculated using the following formula:
% Growth inhibition = (Mean OD of normal control - Mean OD of test group/Mean OD of Normal control) × 100
[00180] In vitro hepatoprotective activity of C. forskohlii (FORCSLIM™): The hepatoprotective activity of C. forskohlii(FORCSLIM™)was evaluated using well maintained HepG2 cells. Ethanol was used as hepatotoxicant and silymarin was used as a standard positive control. The toxic concentration of ethanol taken was 100 mM. The difference of concentrations of C. forskohlii(FORCSLIM™)and standard was based on the results of the MTT assay. The experimental groups were carried out in triplicate as follows:
[00181] Group I (Control):
[00182] Normal control: The cells were treated with100 µl of serum-free culture medium for 24 hrs.
[00183] Dimethyl sulfoxide (DMSO) control: The cells were treated with 100 µl of serum-free culture medium containing DMSO (0.3% v/v) for 24 hrs.
[00184] Silymarin control: The cells were treated with 100 µl of serum free culture medium containing silymarin (200 µg/ml) for 24 hrs.
[00185] C. forskohlii (FORCSLIM™)control: The cells were treated with 100 µl of serum-free culture medium containing C. forskohlii (Forcslim™) (200 µg/ml) (FORCSLIM™) for 24 hrs.
[00186] Group II (toxin treatment): The cells were treated with 100 µl of serum-free culture medium containing 100 mM ethanol for 24 hrs.
[00187] Group III (silymarin treatment): The cells were treated with 100 µl of serum-free culture medium containing 100 mM ethanol with silymarin at a concentration of 50 and 100 µg/ml for 24 hrs.
[00188] Group IV C. forskohliitreatment (FORCSLIM™) : The cells were treated with 100 µl of serum-free culture medium containing 100 mM ethanol with C. forskohlii(Forcslim™) at a concentration of 25, 50, 100, 200 and 400 µg/ml for 24 hrs. Later, cell viability, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) leakage assays were performed for all groups according to the standard method using Ecoline diagnostic kits.
[00189] Results: In vitro hepatoprotective activity of the extract, C. forskohlii(FORCSLIM™) , the results of the study are shown in Table 13. The present investigation indicates that, the ethanolic extract of C. forskohlii (FORCSLIM™) exhibit hepatoprotective effect against ethanol induced hepatic damage. The extract C. forskohlii(FORCSLIM™) has shown a dose-dependent cytoprotective activity with maximum protection at 400µg/ml. The percentage cell viability of the extract, C. forskohlii(FORCSLIM™) at 400µg/ml was more, i.e., 70.32% which was well comparable to that of standard drug silymarin (100µg/ml). The study revealed that the C. forskohlii(FORCSLIM™) extract had shown significant hepatoprotective activity at all the test doses against ethanol-induced cytotoxity.
Table 13: In vitro hepatoprotective activity of C. forskohlii (FORCSLIM™) using HepG2 cell line
Groups %Cell viability LDH ALT AST
Group I (control) 97.98±1.10 135.33±4.17 9.41±1.13 12.66±1.83
DMSO control (0.1% v/v) 94.68±1.04 143.11±3.38 10.38±1.63 14.14±1.04
Silymarin control (100 µg/ml) 97.84±1.02 140.36±4.13 9.83±1.54 13.12±1.13
C. forskohlii (FORCSLIM™) control (200 µg/ml) 95.11±1.08 138.31±3.72 10.92±2.42 14.03±1.29
Group II (toxin treatment)
100 mM ethanol 31.27±1.02 230.6±5.42 36.38±2.15 49.31±2.54
Group III silymarin treatment
100 mM ethanol + silymarin (50 µg/ml) 78.31±1.81 156.31±3.16 15.17±2.14 20.14±2.17
100 mM ethanol + C. forskohlii(FORCSLIM™) (25 µg/ml) 45.23±1.01 210.3±1.32 33.63±2.43 38.35±1.76
100 mM ethanol + C. forskohlii(FORCSLIM™) (50 µg/ml) 50.15±1.02 200.3±1.2.98 29.62±2.53 33.53±1.32
100 mM ethanol + C. forskohlii(FORCSLIM™) (100 µg/ml) 57.14±1.63 191.341±3.23 28.64±2.64 30.34±2.03
100 mM ethanol + C. forskohlii (200µg/ml) 66.35±2.04 184.62±4.21 23.4±2.63 27.37±2.83
100 mM ethanol + C. forskohlii (400µg/ml) 70.32±2.08 179.27±4.82 20.4±2.93 24.36±2.94

In vitro Approach to Evaluate the Anti Adipogenesis Activity of
Coleus forskohlii (Forcslim™) Root Extract in 3T3-L1 Cell Lines
[00190] The present study was carried out to determine the anti-adipogenic as well as anti-hyperlipidemic activities extract of Coleus forskohlii(Forcslim™) using 3T3-L1 Murine adipocyte cell lines. MTT assay was performed for cell viability studies on 3T3-L1 cell lines using the extract. The anti-hyperlipidemic activity of the Coleus forskohliiin 3T3-L1 cell lines was evaluated by Inhibition of pancreatic lipase and lipid accumulation study using Oil Red O Staining wherein Orlistat were used as the reference standard. The results C. forskohlii (FORCSLIM™) exhibited 25.82% at 200 µg concentration which was higher when compared to Simvastatin whose cell viability was 38.34% at 50 µg. The Lipid accumulation was substantially inhibited by test extract and simvastatin at 49.98% and 33.43% respectively whereas the % inhibition of the pancreatic lipase in matured 3T3-L1 adipocytes by the inhibition of breakdown of triglycerides was found to be 54.82 % and 49.83 % at 400 µg for C. forskohlii(FORCSLIM™) and Orlistat respectively.
[00191] Cell lines and culture medium
[00192] 3T3-L1 cell lines were obtained from National Centre for Cell Sciences (NCCS), Pune, India. Stock cultures of the cell lines were cultured in DMEM medium supplemented with 10% inactivated fetal bovine serum, Penicillin (100 IU/ml), Streptomycin (100 mg/ml), and Amphotericin B (5 mg/ml) in a CO2 atmosphere at 37°C until confluent. ?e cells were dissociated with 0.2% trypsin, 0.02% EDTA in PBS solution. All the experiments were carried out in 96 well microtitre plates (Corning, USA).
[00193] In vitro cell viability of the plant extract in 3T3-L1 cells
[00194] The effect of plant extract on cell viability was performed by MTT assay. For testing the in-vitro cytotoxicity of the plant extract, an MTT assay was performed to assess the cell viability with concentrations of test extract of the selected plants ranging from 25µg to 400µg. 100µl of different test concentrations of test drugs were added on to the partial monolayer in microtitre plates. The plate was incubated for 24 hr at 37°C in a 5% CO2 atmosphere. MTT reagent to a final concentration of 0.5 mg/mL of total volume was added to the wells and further incubated for 3 hours. The MTT reagent was replaced with 100 µl DMSO. Simvastatin was used as a positive control. The absorbance was read using an ELISA reader at 570 nm and 630 nm. The IC50 value was determined by using a linear regression equation. The percentage growth inhibition was calculated using the following formula:
[00195] In vitro anti-obesity studies of Coleus forskohlii(Forcslim™) in 3T3-L1 cells:
[00196] Pancreatic lipase Inhibition Assay
[00197] The pancreatic lipase inhibitory activity of given sample is determined using p-nitrophenyl palmitate (pNPP) as a substrate. The enzyme under the reaction conditions hydrolyses p-NPP to release pnitrophenol, which is a coloured substance and can be monitored at 410 nm. The cells were exposed to various concentrations of the crude extracts (25to 400µg) and fractions were prepared in DMSO (25-400) µg/mL. Lipase (0.1 mg) was dissolved in 7ris-bu??er (50 mM, pH 8) and added to the cell supernatant. The mixture was stirred for 15 min and centrifuged at 2000 rpm for 10 min. The clear supernatant was recovered. Different concentrations of given sample (or, Orlistat) was mixed with 0.5 mL lipase solution. It was incubated for 30 min at 37°C. Then, 1 mL substrate p-NPP (3 mM in 2-propanol) was added to all the tubes. After incubating the mixture for 2 hr at 37°C, its absorbance was recorded at 410 nm against a blank. The control contained all constituents except a test sample. Orlistat was used as a positive control.
[00198] The percent inhibition was calculated using the following formula:
% of Lipase activity=100 A sample- A Blank
A control - A Blank
Where, AS and AB are the absorbance of Sample and Control, respectively

[00199] In vitro anti-adipogenic studies of Coleus forskohlii (FORCSLIM™) in 3T3-L1 cells:
[00200] Oil red O staining method the test concentrations of plant extract for anti-adipogenic studies were determined based on the results obtained in cytotoxicity studies and test were carried out on 3T3-L1 cells and the effect of plant extracts on inhibition of fat droplet formation was determined by quantification of Oil Red O staining method.
[00201] Lipid (Oil red O) staining: 1000 µl cell suspension was seeded in a 6- well plate at required cell density (106 cells per well), without the test agent. The cells were allowed to grow for about 72 hr. A stock solution of 1000 mcg/mL was prepared by making up the sample extract and DMEM supplemented with 2% inactivated FBS concentration. The plate was incubated for 24 hr at 37°C in 5% CO2 atmosphere, following which the spent medium was removed.
[00202] Cell Fixing was done on the removal of the cells from the media by gentle washing with PBS. 10% formalin was added to each well, incubated for 30 min to 1 hr. Cell Staining was performed based on the standard procedure. The absorbance was read on an ELISA reader at 492 nm.
[00203] The Oil Red O intensity in treated samples was calculated relative to untreated samples using the following equation:
% Intensity of oil Red Staining= Absorbance of treatment x 100
Absorbance of control

[00204] Results
[00205] In vitro anti-obesity studies of Coleus forkohlii in 3T3-L1 cells:
[00206] The aim of pancreatic lipase activity assay was to know the ability of plant extract to inhibit lipid absorption into the body by inhibiting pancreatic lipase activity. The two main products formed by the hydrolysis of pancreatic lipase are fatty acid and 2-monoacylglycerol. Pancreatic lipase activity is known to act by promoting absorption of monoglyceride and free fatty acid into the body, which is a known cause of obesity. Due to inhibition of pancreatic lipase activity of C. forkohlii extract prevents lipid accumulation into the body.Orlistat was chosen as the standard drug of comparison, approved by FDA and available for the obesity treatment apart from centrally acting antiobesity drugs. This extract acts through the pancreatic lipase inhibition. The untreated cell lines showed 0% lipase inhibition activity; the Orlistat showed 49.83% lipase inhibition activity and the extract showed a dose dependent increase in the inhibition activity i.e, 11.68% lipase inhibition activity at 50 µg and 54.82% lipase inhibition activity was reported at 400 µg respectively ( Table 14 and 15).
[00207] FIG. 3 shows the images for MTT cell viability assay at 20X, according to an embodiment of the present invention.
Table 14: MTT Cell viability readings; Data is shown as Mean ± SEM where n=3 and * shows p value <0.05.

Absorbance (mean ± SEM) Cell viability (%)
Blank 0.07
Control group 0.5634±0.0042 100±0.002
Simvastatin (50 µg)) 0.2664±0.004 38.34±0.003
C. forkohlii25µg/ml 0.4673±0.003 70.53± 0.032
C. forkohlii50µg/ml 0.3863±0.006 52.26±0.004
C. forkohlii100µg/ml 0.2896±0.005 40.62±0.006
C. forkohlii200µg/ml 0.1983±0.007 25.82±0.002
C. forkohlii400µg/ml 0.1297±0.08 16.42±0.089

Table 15: The inhibitory effect of different concentrations of Coleus forkohlii on pancreatic lipase enzyme

Absorbance (mean ± SEM) Lipase inhibition activity (%)
Blank 0.0013
Control group 0.646 ± 0.0153 100.00 ± 0.01
Standard group (Orlistat50 µg) 0.3163 ± 0.0012 49.83 ± 0.002
C. forkohlii25µg/ml 0.5435 ± 0.0042 11.68 ± 0.002
C. forkohlii50µg/ml 0.4963 ± 0.0010 22.54± 0.001
C. forkohlii100µg/ml 0.4183 ± 0.0056 33.64 ± 0.002
C. forkohlii200µg/ml 0.3482 ± 0.0063 42.43 ± 0.002
C. forkohlii400µg/ml 0.2642 ± 0.0073 54.82 ± 0.001

Table 16: Oil Red O staining values on 3T3-L1 cell line; Data is recorded for single concentrations only after MTT assay and shown as Mean ± SEM where n=3 and * shows p-value <0.05
Control Simvastatin (50 µg)) Test compounds
Reading 1 0.778 0.276 0.367
Reading 2 0.807 0.246 0.374
Reading 3 0.811 0.249 0.378
Mean± 0.798±0.0002 0.257±0.0005 0.373±0.0002
% intensity 100 33.43±0.0003 49.98±0.005

In Vitro Antidiabetic Activity C. Forskohlii Root (Forcslim™)
[00208] This study was aimed at assessment of in vitro antidiabetic activities of ethanolic extract of C. forskohlii root (FORCSLIM™) by using 3T3L1 cell line. The cytotoxic effect of the root extract was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The glucose uptake–inducing capabilities and its correlation with glucose transporter 4 (GLUT4) translocation were measured by flow cytometry in 3T3L1 cells. In addition, the inhibitory effect of C. forskohlii root extract(FORCSLIM™) ON a-amylase activity was determined by colorimetric methods. Different concentrations of C. forskohlii root extract(FORCSLIM™) DID not show any toxicity on 3T3L1 cells, after the treatment for 24h. On stimulation with root extract, 62.34% and 76.86% of 3T3L1 cells showed glucose uptake and GLUT4 expression, respectively. The colorimetric assays showed that the ethanolic leaf extract of C. forskohlii root(FORCSLIM™) has a significant inhibitory effect on the activity of a-amylase enzyme inhibitory concentration (IC50) value of 238.65 µg/mL.On the basis of the results of this study, it is evident that C. forskohlii root extract (FORCSLIM™) showed promising anti-diabetic effect when compared to the standard drugs metformin and acarbose and was nontoxic to 3T3L1 cells. Thus, it can be further investigated to recommend as a possible alternative treatment in antidiabetic applications.
[00209] Material and methods
[00210] Cell culture
[00211] 3T3L1 cell line was obtained from the National Centre for Cell Science (NCCS), Pune, Maharashtra, India, and cultured in DMEM (high glucose) supplemented with 10% FBS, 10,000 units of penicillin G, 10,000 µg/mL streptomycin sulfate, and 10mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) at 37°C in 5% CO2 atmosphere.
[00212] Cytotoxicity assay:The cytotoxicity of leaf extract of C. forskohlii(FORCSLIM™) was determined by MTT assay. The yellow-colored tetrazolium salt, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is converted to formazan crystals by the action of lactate dehydrogenase enzyme, produced by live cell mitochondria. 3T3L1 cells at a density of 20 × 104 cells per well/200 µL were seeded in a 96-well plate and cultured overnight. The spent medium was replaced with different concentrations of leaf extract (25–400 µg/mL) diluted in DMEM, and incubated for 24h at 5% CO2, and 37°C temperature. After 24h of incubation, the cells were treated with 0.5mg/mL of MTT reagent and incubated at 37°C temperature for 2h. Further, MTT reagent was removed and the formed formazan crystals were dissolved by adding 20 µL of DMSO. The purple-colored solution was measured at 570nm by microplate reader. The percentage of cell viability was calculated by considering the untreated cells as 100% viable population and using the following formula:

[00213] Glucose uptake assay:In a six-well plate, 3T3L1 cells were seeded at a density of 2 × 105 cells/2mL and incubated in 5% CO2 overnight at 37°C. Later, the spent medium was removed and the cells were washed with D-PBS and treated with experimental compounds and controls in 2mL glucose-free culture medium containing 100 µM 2-NBDG and incubated for 2h. At the end of the treatment, medium was removed from all the wells and washed with D-PBS. Cells were harvested by trypsinization and washed with D-PBS, followed by centrifugation for 5min at 300 × g at 25°C. The supernatant was aspirated and the cells were resuspended in 0.5mL of D-PBS. FACSCalibur, BD Biosciences, USA was used to analyze the cellular uptake of 2-NBDG, by measuring fluorescence intensity in FL1 channel, and CellQuest Pro software, BD Biosciences, USA was used for data analysis.
[00214] Results
[00215] Cytotoxicity effect of C. forskohlii(FORCSLIM™) on 3T3L1 cell line:
The given test compound, C. forskohlii root(FORCSLIM™) extract, at varying concentrations (25–400 µg/mL) did not show cytotoxicity on 3T3L1 cells after the treatment for 24h. The concentrations of C. forskohlii root extract(FORCSLIM™) used to treat the 3T3L1 cells and the respective percentage of viability are given in Table 17. FIG. 4 shows the Cell viability of 3T3L1 cells treated with different concentrations of C. forskohlii(FORCSLIM™) extract and 100 µM of control drug, according to an embodiment of the present invention. FIG. 5 shows images of 3T3L1 cells taken by inverted light microscopy after the exposure to test compounds. (A) Condition medium, (B) Standard metformin drug (100 µM), and (C) 400 µg/mL of C. forskohlii root (FORCSLIM™) extract treated cells for 24h, according to the embodiment of the present invention. As metformin is a standard antidiabetic drug and has been used to conduct in vitro cell-based antidiabetic activities, the toxicity of metformin was also tested against 3T3L1 cells and it showed 91.52% of viability at 100 µM concentration. Untreated cells were considered as 100% viable cells. Each experiment was performed thrice and the results are represented as mean values ± SD [Table 17].
[00216] Glucose uptake assay
[00217] 2 NBDG, a fluorescent deoxyglucoseanalogue, was used to probe for the cellular uptake of glucose in 3T3L1 cells. The results showed that the amount of cells that took up 2 NBDG were higher in the population of cells treated with C. forskohliiextract when compared to the untreated cells. Metformin treated cells showed the highest cellular uptake of 2 NBDG. The relative mean fluorescence intensity values are given in FIG. 6 and 7. FIG. 6 shows overlaid expression graph for the presence of fluorescent 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) in given untreated 3T3L1 cells (black color line) and standard drug–treated cells (metformin, 100µM) (red color line) and 100 µg/mL of C. forskohlii root extract(FORCSLIM™)treated cells (green color line), according to the embodiments of the present invention. FIG. 7 shows overlaid fluorescence intensities of given untreated 3T3L1 cells (Black colour line) and Standard drug treated cells (Metformin 100µM, Red colour line) and 100µg/mL of C. forskohlii extract(FORCSLIM™)treated cells (Green Colour line), according to an embodiment of the present invention.
[00218] Glucose transporter 4 (GLUT4) expression study on 3T3L1 cell line by flow cytometry: The observation in statistical data of GLUT4 expression study by flow cytometry suggests that 0.23% of cells in untreated population, 99.74% cells in metformin (100 µM) treated population, and 76.86% of cells in C. forskohlii root extract(FORCSLIM™)treated population showed GLUT4 expression. The results are shown in FIG. 8.FIG. 8 shows glucose transporter 4 (GLUT4) expression study on 3T3L1 cell line by flow cytometry, according to an embodiment of the present invention.
Table- 17 1. Cell viability effects of C. forskohlii extract(FORCSLIM™) in 3T3L1 cell line
Culture conditions Percentage of cell viability (%)
Control 100.00±00
25 98.21±1.14
50 97.12±1.17
100 93.42±1.32
200 91.45±1.54
400 90.78±1.58
Metformin(100µM) 91.46±1,63

G) ADVANTAGES OF INVENTION
[00219] The present inventionprovides extract of Coleus forskohlii(Forcslim™) free from 14-deoxycoleon U compound that is the main cause of fatty liver. The present invention prevents induced hepatotoxicity, thus making Coleus forskohlii (Forcslim™) extract completely safe for consumption. A complete elimination of 14-deoxycoleon U from the Coleus forskohlii(Forcslim™) extract makes the composition safe to consume. Hence, theColeus forskohlii(Forcslim™) extract of present invention is used in treating obesity, cardiovascular disorders, adenocarcinoma etc. even for a long period of time without any side effect.
[00220] It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the claims presented in the complete specification or non-provisional application.