In vitro studies
Cell culture and treatment
Mouse pre-adipocyte fibroblasts 3T3-L1 cells were obtained from American Type Culture Collection (Manassas, VA) and cultivated in maintenance medium comprised of DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate and 4.5 g/L D-glucose.
Equal number of 3T3-L1 cells (60,000 cells per well) was seeded in each well of 24-well culture plates. Cells were pre-treated with different concentrations of herbal extracts or formulations for 2h and incubated further with the differentiation medium containing 500 nM insulin, 1 μM dexamethasone, and 0.5 mM IBMX for 48h. Thereafter, cells were maintained in the post-differentiation medium (DMEM containing 100nM insulin) in presence or absence of LI10903F for further 8 days. The control cultures received only 0.1% (v/v) DMSO as the vehicle. The intracellular lipid accumulation was measured by staining the cells with Oil Red O stain following the method described earlier.
The intracellular lipid breakdown efficacy of herbal extracts and their formulations was evaluated by measuring the released glycerol in the 3T3-L1 culture supernatants. Equal number of mature adipocytes was treated with different concentrations of either P. Betle or D. biflorus extracts or their formulations in phenol red-free DMEM supplemented with 2% bovine serum albumin (BSA) for 4h. Released glycerol in the cell free culture supernatants was measured using the Adipolysis Assay Kit (Millipore, Billerica, MA) as described previously.
Acute oral and 28-day dose-dependent oral toxicity studies were conducted at Laila Impex R&D Centre (Vijayawada, India). In vitro chromosome aberration test and mammalian erythrocyte micronucleus test were conducted at Shriram Institute for Industrial Research (Delhi, India). Ames bacterial reverse mutation assay was conducted at Vimta Labs Limited (Hyderabad, India). All studies complied with the Good Laboratory Practice (GLP) Standards, and were done according to the OECD guidelines for the testing of chemicals. The details of these procedures were described previously by Sreejayan et al..
Acute oral toxicity
An acute oral toxicity study was conducted following the protocol as described earlier. Three Sprague–Dawley (SD) female rats, nulliparous and non-pregnant (age: 12 weeks; 205–230 g body weight at study initiation) were administered orally with a single dose of 5000 mg/kg of LI10903F. All the animals were observed for mortality, signs of gross toxicity, and behavioral changes on the day of dosing and thereafter for 14 days. All animals were euthanized using anesthetic ether at the end of the observation period and subjected to necropsy.
28-day sub-chronic toxicity
A twenty-eight day sub chronic toxicity study was conducted following the protocol as described earlier. Forty healthy Sprague Dawley rats (20 males and 20 females) were equally divided into four groups. Each group of animals (n=10) was supplemented with 50 or 250 or 2500 mg/kg LI10903F or vehicle (1% CMC). The animals were given LI10903F for 28 consecutive days by oral gavage using a ball-tipped gavage needle attached to an appropriate syringe. Animals were monitored for mortality, toxicity, and behavioral changes throughout the study period. Body weights were recorded before dosing (Day 0), and at weekly intervals thereafter.
Ames test: reverse mutation assay
The reverse mutation test measures the ability of LI10903F to induce reverse mutation at selected histidine loci in five tester strains of Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535, and TA 1537 (Molecular Toxicology, Boone, NC). In the dose-range finding study, the herbal blend was tested at the dose levels of 39.06, 78.13, 156.25, 312.50, 625, 1250, 2500, and 5000μg/mL in presence (10% S9 fraction v/v) or absence of metabolic activation system.
Mammalian erythrocyte micronucleus test
A total of 60 healthy Swiss albino mice (30 males, 30 females) of approximately 8–12 weeks age were randomly assigned to three main study groups (n=20) with equal number of either gender of animals in each group. Animals in Group 1 were administered orally with 2000 mg/kg of LI10903F; animals in Group 2 and Group 3 were administered with corn oil (negative control) and 40 mg/kg cyclophosphamide via i.p., respectively. Animals were observed for toxicity or mortality. Five males and five females from each group were sacrificed after 24h, and similarly five males and five females after 48h of dose administration. The bone marrow smears were prepared from femur, stained with May and Graunwald stain and examined for polychromatic erythrocytes (PCE) under 40X objective of a Nikon Eclipse TS 100 microscope (Nikon Corporation, Japan).
Mammalian chromosome aberration test
Whole blood was collected in heparinized vial from two healthy male human volunteers. The test was conducted on isolated lymphocytes at 1000, 2500 and 5000 μg/mL of LI10903F with or without metabolic activation. Cultures treated with mitomycin-C or cyclophosphamide served as positive control, whereas, culture treated with 0.1% DMSO as negative control. Colchicine was added to the cultures 2h prior to harvesting. After 18h of treatment with LI10903F, lymphocytes were harvested for assessing chromosomal aberration. Metaphase preparations were stained with Giemsa and aberrations classified and scored under 40X objective of a Nikon Eclipse TS 100 microscope (Nikon Corporation, Japan).
Clinical study material
The study material LI10903F or LOWAT is a blend of 60% alcohol extract of P. bettle leaves standardized to 6% polyphenols plus 90% alcohol extract of D. biflorus seeds standardized to 4% γ-glutamyl phenylalanine mixed in 2:3 ratio. For clinical study, three hundred milligrams of LI10903F was encapsulated in opaque capsules with 200 mg of exicipient. A combination of micro crystalline cellulose and Magnesium stearate (<2%) was used as the excipient. Placebo capsules contained only exicipient, and were identical in appearance, size, weight and color to the active capsules. The formulation was provided by InterHealth Nutraceuticals (Benicia, CA).
Trial site and recruitment of subjects
This randomized, double-blind, placebo controlled, clinical trial (RCT) was performed at Alluri Sitarama Raju Academy of Medical Sciences (ASRAM), Eluru, Andhra Pradesh, India (clinical trial registration number: ISRCTN37381706). The protocol was evaluated and approved by the ASRAM Institutional Review Board (IRB). IRB approval Reference number, 07-001/IB/PHF OB.
One hundred forty five subjects participated in a questionnaire-based screening process. Inclusion criteria were: adults (21–50 years), BMI ≥ 30 kg/m2, female participants should not be pregnant, willingness to follow method of birth control if premenopausal. Exclusion criteria were: morbidly obese (BMI > 40 kg/m2), history of thyroid disease, cardiovascular disease, diabetes, hepatitis, pancreatitis, lactic acidosis or hepatomegaly with steatosis, motor weakness, peripheral sensory neuropathy; allergies to spices and herbal products; using weight loss medications, laxatives or diuretics taken solely for the purpose of weight loss; recent unexplained weight loss or gain; HIV positive; any evidence of organ dysfunction or any clinically significant deviation from normal that could impact subjects well-being or clinical outcome. Of 145 candidates screened 50 subjects who met eligibility criteria were enrolled in the study. Each participant voluntarily signed the IRB-approved, informed-consent form. At baseline visit, the subjects were randomized into either the placebo or LI10903F group.
At baseline visit, all subjects were provided with LI10903F or placebo capsules, compliance cards, and dates for follow-up evaluations. A standard diet (2,000 kcal daily) was provided (breakfast, lunch and dinner) deriving 61% kcal from carbohydrate, 14% from protein and 25% from fat. The subjects were required to walk 30 min, five days a week under the supervision of a physical trainer. The subjects were instructed to take 3 capsules a day, one each 30 min before breakfast, lunch and dinner. All subjects completed a medical history questionnaire at baseline and compliance reports during follow-up evaluations at 14, 28 and 56 days. Subjects were assessed for body weight, height, waist plus hip circumference and vital signs at baseline and subsequent follow-up visits. At all study visits, blood samples were collected for hematological and biochemical evaluations, and urine samples for urinalysis. Reduction in body weight and BMI were considered as the primary study outcome parameters of the study. Sample size calculations were done using power analysis based on previous obesity study report. We estimated that more than 95% power at the two-tailed α level of 0.05 would be provided to test the significance of weight reduction over placebo with 25 subjects per group.
Serum adipokine assay
Serum adiponectin and ghrelin levels were determined using the human Adiponectin ELISA kit (Millipore, St. Charles, MO) and the human Ghrelin ELISA kit (Millipore, St. Charles, MO). The detection sensitivity of adiponectin and ghrelin assays was 0.78 ng/ml and 8 pg/ml, respectively.
Mean values were compared with analysis of variance (ANOVA) on variables for between-group differences at each individual time point with repeated measurements using group and time as main effects and baseline of the variables and gender as covariate. For baseline (week 0) comparisons, a t-test was used. For inter group comparisons, a p-value < 0.05 is considered to be statistically significant. All comparisons reported are between the placebo and LI10903F groups at each specified time point. All analyses were performed with SYSTAT (Windows version 11.0; San Jose, CA).