Efficacy of a novel herbal formulation for weight loss demonstrated in a 16-week

randomized, double-blind, placebo-controlled clinical trial with healthy overweight adults.

Short Title: Clinical efficacy of an herbal blend in overweight adults

Accepted Article

Kashinath Dixit M.D.1, Dinesh Venkata Kamath M.D.2, Krishnaraju Venkata Alluri M.S.3,

Barbara A. Davis Ph.D., R.D.4*

1
Krupa Centre for Diabetes and Obesity, Usha Nivas, 1149, Bengaluru - 560086, Karnataka,
India
2
Sudeep Diabetes Care Centre, Malleswaram, Bengaluru - 560003, Karnataka, India
3
Laila Nutraceuticals R&D Center, Vijayawada-520007, India
4
PLT Health Solutions Inc., Morristown, NJ 07960

*Corresponding Author:
Barbara A. Davis, PhD, RD
barbara@plthealth.com
119 Headquarters Plaza, Morristown, NJ 06019
Phone: 973-984-0900 ext. 252

KEY WORDS: Body Mass Index, Body weight management, Curcuma longa, LI85008F,
Moringa oleifera, Murraya koeingii, Randomized Double-Blind Placebo-Controlled Clinical
study

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which
may lead to differences between this version and the Version of Record. Please cite this
article as doi: 10.1111/dom.13443

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 ABSTRACT:

Aims: LI85008F is a novel, proprietary herbal blend containing extracts of Moringa oleifera and

Murraya koeingii leaves plus extract of Curcuma longa root. Weight loss efficacy of LI85008F

was first demonstrated in obese adults and has now been re-evaluated in healthy overweight

adults via a 16-week randomized, double-blind, placebo-controlled clinical study.

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Material & Methods: One hundred and forty overweight participants (Body mass index 27 to

29.9 kg/m2, 29.3% male; ages 21-50 years) were randomized into placebo (n = 70) and LI85008F

(n = 70) groups. The participants received either 900 mg/day of LI85008F in two divided doses

or two identical placebo capsules. In addition, participants were counseled to follow

approximately 1800 kcal/day diet and to engage in walking for 30 min, 5 days/week throughout

the study.

Results: At the end of the trial period, the LI85008F supplemented group showed significant

reductions in body weight (5.36±1.769 vs. 0.87±1.381kg; p<0.0001) and BMI (2.05±0.693 vs.

0.34 ±0.559 kg/m2; p<0.0001), compared with placebo. Significant reductions in waist and hip

circumferences, and a 2.08-fold reduction of waist/hip ratio, were noted in the LI85008F

supplemented group. LI85008F supplementation also resulted in significant improvements in

lipid profiles, compared with the placebo; LDL cholesterol decreased, while HDL cholesterol

increased, resulting in a significantly improved LDL/HDL ratio. No major adverse events were

reported by the participants in the study duration.

Conclusions: The unique herbal extract blend LI85008F, combined with modest calorie

restriction and physical activity, is well-tolerated, safe, and effective for weight management in

overweight men and women.

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 INTRODUCTION

An estimated one billion adults are overweight and at least 300 million are obese worldwide,

with prevalence increasing in most countries (Ogden et al., 2007; WHO Fact Sheet, 2016). Body

mass index (BMI) is an established measure for classifying weight and is often used as a
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surrogate for total body fat (I:\7544dft.doc, 2007). Overweight and obesity, classified by a BMI

of 25–29.9 and ≥ 30 kg/m2, respectively, have been associated with increased risk of

comorbidities such as type-2 diabetes, cardiovascular disease, osteoarthritis, and some cancers

(Whitlock et al., 2009).

Alternative strategies for weight management have become widely adopted because of the high

cost and potential adverse effects of conventional pharmaceutical and/or surgical interventions.

Among those approaches, nutritional ingredients such as botanicals are popular for management

of overweight and obesity (Allison et al., 2001).

The herbal combination, LI85008F, was developed to meet the need for high-quality, clinically

evaluated botanical weight management dietary ingredients. An in vitro screening program,

established by Laila Nutraceuticals (Vijayawada, India), evaluated hundreds of herbal extracts

for their ability to inhibit fat accumulation by adipocyte differentiation (i.e., adipogenesis) and

potentiate fat breakdown within cells (i.e., lipolysis) in 3T3-L1 mouse adipocytes (Sengupta et

al., 2011). The three extracts shown to be most effective in this screening were combined in

various ratios, with the most effective synergistic combination being LI85008F. The resulting

dietary ingredient inhibits lipogenesis in adipocytes while concurrently antagonizing PPARγ and

other lipogenic factors. In addition, it potentiates triglyceride mobilization from fat cells, i.e.,

enhances lipolysis (Sengupta et al., 2011).

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 Supplementation of LI85008F to obese adults in an 8-week double-blind placebo-controlled

clinical study resulted in significant reductions in weight and BMI along with improvements in

serum lipid profiles and a significant increase in serum adiponectin. Observations relevant to

safety parameters supported that LI85008F was well tolerated and safe (Sengupta et al., 2012).

A comprehensive set of in vitro and in vivo toxicological studies also support the safety of
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LI85008F (Alluri et al., 2010).

The clinical efficacy and safety profile of LI85008F prompted further evaluation in a larger

population of overweight participants over a longer duration. This communication presents the

clinical efficacy and tolerability of LI85008F on healthy overweight participants (BMI 27-29.9

kg/m2) in a 16-week double-blind placebo-controlled study.

MATERIALS AND METHODS

Study material

The herbal formulation LI85008F was prepared in the ratio of 6 parts Moringa oleifera leaf

aqueous ethanol extract, 3 parts Murraya koenigii (L.) Spreng. (family Rutaceae) leaf aqueous

ethanol extract and 1 part Curcuma longa L. (family Zingiberaceae) extract standardized to 95%

total curcuminoids. Identification, collection of the plant raw materials and the extraction

procedures of the individual ingredients from the respective raw materials were described

previously (Sengupta et al., 2011, 2012). The finished formulation was standardized to contain at

least 7.0% total curcuminoids, 0.1% Mahanine and 0.2% Quercetin 3-O-glycoside. LI85008F

was produced in a cGMP certified manufacturing facility at Laila Nutraceuticals, Vijayawada,

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 India. This formulation is available commercially as Slimvance®/Slendacor® through PLT

Health Solutions, Morristown, NJ.

A typical HPLC chromatogram of this ingredient is presented in Figure 1. For analysis,

LI85008F was solubilized and diluted in 0.1% Orthophosphoric acid: Methanol (20:80) and

analyzed using a HPLC system connected to a PDA detector equipped with Empower3 software
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(Waters Alliance Corp., Milford, MA). The sample was applied onto an X Bridge C18 column

(100 x 4.6mm, 3.5 μm) (Waters Corp., Milford, MA, USA) through a 10μl auto injector. The

mobile phase consisted of a gradient of 0.1% Orthophosphoric acid: Acetonitrile. The run was

conducted at 30oC for 32 min and the eluted samples were analyzed at 254nm. Analysis of the

final formulation i.e. LI85008F was carried out in the Analytical department of Laila

Nutraceuticals, Vijayawada, India. In addition, possible contaminants such as microbial and

heavy metals were also analyzed.

The dose of LI85008F (900 mg/day) in the clinical study was chosen based on preclinical proof

of concept study conducted in high fat diet induced obese rats (unpublished observation). For

clinical study, LI85008F was encapsulated in size zero hard gelatin capsules; the placebo

capsules contained 99.56% maize starch and 0.44% syloid. The capsules containing LI85008F or

placebo were practically indistinguishable, being identical in size, weight, and external

appearance such as color and texture. At each visit, all study product was collected, and unused

capsules were counted to determine compliance. Missed doses were captured in the Daily Diary

and Compliance Card and in the respective CRF pages.

Study Objectives & Outcomes

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 The purpose of the present clinical investigation was to evaluate the weight loss efficacy and

tolerability of LI85008F in healthy overweight participants. The primary outcome at the end of

treatment period was change in body weight from baseline. Secondary outcome measures

included reduction in BMI, waist and hip circumference, changes in body composition (reduced

fat mass), serum lipid, adiponectin, and ghrelin profiles. Vital signs, ECG, clinical chemistry,
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hematology and reported adverse events were considered as parameters to evaluate the safety

and tolerability.

Screening and Recruitment

The clinical study protocol was reviewed and approved by an ethics committee (Bangalore

Ethics, Bengaluru, India); the approved protocol was registered (Clinical Trial Registry-

India/2015/06/005835). The study was conducted in accordance with the ethical principles of the

Declaration of Helsinki and are consistent with Good Clinical Practice and the applicable

regulatory requirements. The study, which began 1 June 2015 and ended 15 Nov 2015, was

conducted at two independent centers - Sudeep Diabetes Care Centre, Bengaluru and Krupa

Centre for Diabetes and Obesity, Bengaluru. These two sites were monitored by an independent

site monitoring organization (SMO). An independent third-party auditor audited technical and

regulatory aspects of the study. The case report forms (CRFs) were audited for compliance to

source data and the trial master file was reviewed for regulatory compliance.

Participants visiting the outpatient departments of the clinics were selected for screening. After

voluntarily signing informed consent, male and female participants were screened for eligibility

of enrollment into the study based on inclusion and exclusion criteria, as described previously

with some modifications (Sengupta et al., 2012). Briefly, criteria for eligibility were that

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 participants be between 21-50 years, BMI 27-29.9 kg/m2, have an apparently healthy status

based on medical history, be free from metabolic bone disease, gastrointestinal disease, diabetes

mellitus (type I or type II), cardiovascular disease, renal disease, abnormal liver function, on no

medications or vitamin supplements, not pregnant or lactating. We excluded individuals with

HIV, inflammatory disorders, unexplained weight loss or gain within last three months, those
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following weight-loss programs, taking weight loss drugs, and those smoking, or drinking

alcohol. Of the 157 participants screened, 140 met the above inclusion criteria and were recruited

for the study.

Study Design:

Participant disposition throughout the study is presented in the CONSORT (Consolidated

Standards of Reporting Trials) flow diagram, Figure 2 (Schulz et al., 2010). Participants

(n=140) were randomized into two groups using a computer-generated block randomization

method (http://www.randomization.com) by an independent statistician and included LI85008F

and placebo groups (n=70/group). The study investigators were blinded to allocation. The

eligible participants were randomized as per the randomization codes labeled on Investigational

product in a 1:1 ratio. Study capsules, compliance card, list of instructions and dates of follow-

up evaluations were provided to all participants at the baseline visit. Each active and placebo

capsule contained 450 mg of LI85008F or 450 mg of excipients, respectively. Participants in

both groups were instructed to take 2 capsules per day; one before breakfast and one before

dinner. They were also advised to walk for 30 minutes 5 days/week and to maintain a diet of

approximately 1800 kcal/day as per instruction by the principal investigator and a dietitian. A

study dietitian shared daily menus with each subject and provided counseling to assist

participants in maintaining the standard diet during the study. The diary card containing

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 information about compliance to the study supplement, diet and exercise were reviewed at each

follow up visit. Study supplement compliance was at least 80%.

The intervention was conducted for 16 weeks, which included baseline and follow up visits at 2,

4, 8, 12 and 16 weeks. At all visits, participants were assessed for anthropometric parameters

including body weight, height, waist circumference, hip circumference, along with vital signs.
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Participants also completed Visual Analog Scale (VAS) appetite scores and Profile of Mood

States - Short Form (POMS-SF) questionnaires. The primary efficacy parameter, body weight,

was measured using an electronic weighing scale (Aliston AL650 Weighing Scale, Mumbai,

India) at all study visits. Body fat assessment was carried out at baseline (visit 2) and week 16

(visit 7) through Dual-energy X-ray absorptiometry (DEXA) (encore-based X-ray Bone

Densitometer; Lunar Prodigy Series, GE Medical Systems).

A copy of the protocol is available upon request to PLT Health Solutions (NJ, USA).

Hematological and biochemical evaluations

For assessment of LI85008F safety, several parameters were evaluated in serum, urine and whole

blood of all participants at the baseline and final visit. Serum lipids were also evaluated.

Biochemical and hematological parameters were measured using the automated analyzer

COBAS INTEGRA (400 PLUS) auto clinical chemistry analyzer, Roche Diagnostics Ltd

(Rotkreuz, Switzerland) and the hematological counter MINDRAY (BC - 5380) auto hematology

analyzer (Shenzhen, China), respectively. Urine analysis was carried out using UroColor Strips

(Standard Diagnostics, Kyonggi-do, Korea) and by microscopy of sediment.

Serum Biomarkers

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 Serum adiponectin and ghrelin levels were determined by specific EIA method using human

ELISA kits, procured from R&D Systems (Minneapolis, MN) and RayBiotech Inc. (Norcross,

GA), respectively, according to the instructions provided by the manufacturers.

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Statistical Analysis

Descriptive statistics are presented as mean±SD. All outcome measures (i.e. primary &

secondary end points) in the per-protocol population were analyzed by parametric tests including

paired t-test and unpaired t-test, to compare means within and between the groups. Comparison

between body weights at the follow up visits between the LI85008F and placebo groups were

analyzed using Analysis of Covariance (ANCOVA).

Analysis of Covariance (ANCOVA) was used to compare inter and intra group reduction of body

weight from baseline to the final visit (i.e. weeks 16) between the LI85008F and placebo groups.

The interaction between the groups over time was evaluated by ANCOVA with the statistical

significance level set at p≤0.05.

Group size estimations were based on power calculations, predicting the effect size and

variations between and within groups. The effect and deviation sizes observed in the previous

clinical study (Sengupta et al., 2012) conduced in obese participants were 1.79 and 1.52 in

placebo; 4.76 and 2.3 in LI85008F groups respectively. As the current study was conducted in

healthy overweight participants, we considered a lower effect size. The assumed effect and

deviation sizes 1.4 and 1.8 in placebo; 3.0 and 2.8 in the treatment groups with sample sizes of

60 per arm were sufficient to achieve a power of >90%. In this study population, we anticipated

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 that there could be at least a 5% reduction of the baseline body weight in the LI85008F group at

the end of the study and estimated a 15% dropout rate. Considering these assumptions, 60

completers per cohort were sufficient to achieve at least 90% power to detect outcome

differences of change in body weight between the groups, assuming a two-sided significance

level of 0.05.
Accepted Article

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 Results:

Demographic and Baseline characteristics

The demographic variables and baseline characteristics are summarized in Table 1. Participants

were randomly distributed into placebo and treatment groups. Overall, the active group receiving

LI85008F (900 mg/day, n = 70) and placebo (n = 70) were not statistically different at baseline
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with respect to any characteristic measured.

Clinical efficacy

Changes in anthropometric measures: Changes from baseline body weight, BMI, waist, and

hip circumferences in both groups are summarized in Table 2. LI85008F supplementation for

16 weeks resulted in statistically significant body weight reduction vs. placebo (5.36±1.769 kg

vs. 0.87±1.381 kg; p<0.0001). Significant reduction in body weight was observed as early as 2

weeks in supplemented participants, when compared with the placebo (0.76±0.365 kg vs.

0.19±0.305 kg; p<0.0001). Average baseline body weight loss in the LI85008F supplemented

group was 7.08%, whereas the placebo group lost only 1.16% of the baseline body weight

(Table 2, Figure 3). LI85008F supplementation for 16 weeks also demonstrated significant

reduction of BMI from baseline vs. placebo (2.05±0.693 vs. 0.34±0.559; p<0.0001) (Table 2).

This statistically significant BMI reduction in LI85008F supplemented group also began as early

as 2 weeks (0.25±0.125 vs. 0.07±0.125; p<0.0001). Furthermore, at the end of the trial period,

LI85008F supplementation resulted in significant reductions in waist (5.38±1.602 vs.

1.70±1.827cm; p<0.0001) and hip (4.49±1.664 vs. 1.19±1.465cm; p<0.0001) circumferences,

when compared with the placebo. The mean reduction of waist/hip ratio in the LI85008F group

was 2.08-fold, compared with the placebo (0.0123±0.020 vs. 0.0059±0.017; p=0.0547) (Table

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 2). Subgroup analyses showed similar, statistically significant improvements in anthropometric

measures for male and female participants (data not shown).

As a secondary outcome measure, body composition using DEXA was also evaluated. At the end

of the trial period, the LI85008F-supplemented group had a reduction of 1.11±7.059 kg body fat;

whereas, participants in the placebo group gained 0.61±5.052 kg body fat from baseline
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(P=0.1151), respectively (Table 3). Of note, there was a statistically significant difference

(P=0.0061) between mean change from baseline lean mass between the LI85008F-supplemented

vs. placebo group. Whereas lean mass was preserved (increased 0.9+3.743 kg) in the

supplemented group, lean mass was lost in the placebo group (-0.91+3.621 kg, Table 3).

Improvement in serum lipid profile: At the end of the 16-week trial period, the herbal

supplementation significantly decreased baseline serum LDL (30.20±18.387 vs. 1.08±9.519

mg/dL; p<0.0001), VLDL (6.80±6.369 vs. 1.09±3.059 mg/dL; p<0.0001), total cholesterol

(28.04±21.805 vs. 5.37±10.203 mg/dL; p<0.0001) and triglyceride (34.12±31.682 vs.

5.09±14.99 mg/dL; p<0.0001) compared with the placebo group (Table 4). Significant

reductions in LDL (p<0.0001), VLDL (p<0.0001), total cholesterol (P<0.0001) and triglyceride

(P<0.0001) were observed as early as 4 weeks. Furthermore, at the end of the study LI85008F

group also showed significant improvement (p<0.0001) in serum HDL level by 8.66±8.908

mg/dl; whereas, in the placebo group HDL level was decreased by 3.51±5.543 mg/dL from the

baseline. The LI85008F-supplemented cohort also showed significant improvement in HDL

(p<0.0012) as early as 4 weeks. At 16 weeks, a notable effect on LDL/HDL ratio (1.37±0.656 vs.

2.25±0.718; p<0.0001) was observed in the LI85008F group compared with the placebo; this

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 effect was also evident as early as week 4 of the study (1.84±0.756 vs. 2.15±0.681; p<0.0133)

(Table 4)

Modulation of adiponectin and ghrelin levels in serum: At the end of the study LI85008F

supplementation resulted in a 34.77% (p = 0.0071) increase in serum adiponectin concentration

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from the baseline (4.279±2.105 vs. 3.175±1.772 µg/ml); whereas, the placebo group showed a

15.05% increase (p=0.1127) in adiponectin level from the baseline (Figure 4A). The LI85008F

group showed 20.17% (p=0.0568) reduction of serum ghrelin levels from the baseline

(97.15±35.42 vs. 121.7±92.74 ng/ml); in comparison, the placebo group showed a slight

reduction of 10.42% (110.46±47.99 vs.123.31±91.48 ng/ml; p=0.1945) (Figure 4B).

Profile of mood states: Overall mood, assessed through the Profile of Mood States (POMS)

questionnaire, was significantly improved in the LI85008F group (p=0.0021, data not shown).

Adverse events and dropouts: No serious adverse events were observed in this study. Minor

events reported by study participants include fever, gastritis, headache, itching, loose stool,

acidity, excessive appetite, and dehydration. These minor adverse events were evenly distributed

among the two groups and probably not related to the study supplement.

Four participants from the LI85008F and six participants from the placebo group were excluded

because of their non-availability during the entire study duration; they were dropped from the

study prior to the outcome measure assessments. Therefore, the number of completers were,

LI85008F (n=66) and placebo (n=64). However, no subject was dropped out of the study

because of an adverse event.

Discussion:

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 Obesity and overweight continue to negatively impact society, overwhelming health, and

healthcare services globally. Despite a real need, relatively few botanical options that are

supported by rigorous scientific research exist to support weight management (Rios-Hoyo,

2016). The present study shows 16-week supplementation of a novel herbal formula (LI85008F)

reduces body weight, BMI, body fat, waist and hip circumference and waist/hip ratio in
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overweight participants with an average BMI of 28.56 kg/m2. In addition, biochemical,

hematological parameters and subject compliance demonstrate that LI85008F is tolerable and

safe for human consumption. Previously, LI85008F was shown to be a clinically effective and

safe herbal composition for weight management in obese participants (Sengupta et al., 2012).

The earlier, 8-week double-blind placebo-controlled clinical study, conducted on a smaller group

(n=41) of obese participants (BMI 30 to 40 kg/m2) demonstrated that LI85008F significantly

reduced the baseline body weight and BMI along with maintaining healthy serum lipid profiles

(Sengupta et al., 2012). Demonstrating the mechanism of action of LI85008F at the cellular

level, another study showed that this novel composition made from three herbal extracts,

synergistically reduced adipogenesis and increased intracellular fat lysis processes in 3T3-L1

adipocytes through a PPAR dependent pathway (Sengupta et al., 2011).

The primary outcome of the present study was reduction of baseline body weight in the active

cohort. Body weight reductions of >5% have been associated with improvements in metabolic

and cardiovascular health (Douketis et al., 2005). Our data show that an average of 7.08% of the

baseline body weight was lost in the LI85008F group after 16 weeks of intervention. Further

analysis reveals that 54 out of 66 participants in the active group lost more than 5% of their

baseline body weight at the end of the trial (Figure 3). In addition, LI85008F supplementation

showed a series of compelling changes in baseline anthropometric measures of the study

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 participants. Reductions from baseline BMI of the LI85008F group versus the placebo group

were highly significant throughout the study and began as early as week 2. This clinical efficacy

of LI85008F is consistent with an earlier study conducted with obese participants (Sengupta et

al., 2012).

BMI is considered as a marker of global adiposity and is also a reliable marker of visceral
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adiposity as explained by the fact that visceral adipose tissue increases in a quasi-linear manner

with BMI in both sexes (Ferrannini et al., 2008). Waist circumference (WC) and waist-hip ratio

(WHR) are commonly used as surrogate markers of visceral adiposity (Alberti et al., 2005).

Visceral obesity is a significant risk factor for cardiovascular morbidity and mortality; therefore,

increased WC is considered as one of the vital diagnostic criteria for Metabolic Syndrome

(Alberti et al., 2005, 2009). In the present study, LI85008F supplementation conferred

significant reduction of WC and a reduction in WHR (p=0.0547), compared to the placebo. The

LI85008F group experienced a significant reduction from baseline WC as early as 2 weeks,

compared to the placebo group. The reduction of visceral adiposity in the LI85008F group is

supported by the observation of a significant reduction of 1.20% total body fat achieved at the

end of the study. Together, these data suggest that LI85008F is an effective option for reducing

body weight and fat in overweight participants.

LI85008F supplemented participants experienced significant improvements in serum markers of

lipid metabolism related to overweight and obesity. Hyperlipidemia is generally associated with

overweight and obesity, which is known as a risk factor for CVD including atherosclerosis

(Kawada 2002; Fischer et al., 2015). The study showed a reduction in serum LDL, VLDL, total

cholesterol, triglyceride and LDL/HDL ratio following a similar pattern to weight loss over the

16-week trial period, with significant effects noticed as early as 4 weeks. Concomitantly, serum

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 HDL level was also significantly improved in the LI85008F group. The reduced level of harmful

lipids in circulation i.e. LDL triglycerides and the LDL/HDL ratio reflected an improved status

of fat metabolism and reduced stored fat in the body. Overall, the improvements in serum lipid

profile in the LI85008F supplemented participants imply possible cardiovascular benefits for

overweight participants.
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In this study, there was a slightly greater percentage of female participants than male

participants. Among the 130 completers, 75 were females (40 participants in LI85008F and 35

participants in placebo group). A chi square test (p=0.4947) suggested that there was no

association between gender variation and treatment effect.

This study, together with the previous investigation (Sengupta, 2012), demonstrates weight loss

efficacy across a range of adult ages, in both genders and in overweight as well as obese

individuals. The fact that not only weight, but body mass index, waist circumference, hip

circumference, waist:hip ratio and fat mass via DEXA were positively modified by LI85008F

suggests consistent efficacy across multiple parameters related to weight management.

Additional research into whether race and ethnicity play a role in efficacy may be warranted.

However, whereas differences in visceral adiposity at various BMIs has been noted across

different ethnic backgrounds (WHO, 2008), this may be more relevant to health outcomes

associated with BMI rather than the efficacy of a weight management ingredient.

A notable limitation of the study is the lack of food intake data. Whereas participants in both

groups were provided with regular counseling and example menu plans by a study dietitian,

dietary intake was not monitored. Accuracy and reliability of food intake assessments is

complex, often requiring multiple assessment tools to collect a true representation of food

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 consumption (Lombard et al, 2015). In a long-term study, such as this 16-week intervention

trial, it placed burden on study participants, especially when the primary outcome measure was

to evaluate efficacy of the dietary ingredient. Participants were required to complete compliance

cards daily and analysis showed no significant difference in diet compliant days between placebo

and treatment groups (data not shown). However, future research could include a design that
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allows us to investigate the impact of dietary intake in conjunction with LI85008F intake.

Together with previous work, the present investigation has established that LI85008F is well-

tolerated, safe, and effective. The individual ingredients of LI85008F have a very long history of

use in the cuisine of Indian and other cultures. Moreover, a preclinical safety study and a clinical

study on obese participants had already established that LI85008F was safe and tolerable for

human use (Alluri et al., 2010; Sengupta et al., 2012). Similarly, in the present study, no major

adverse events were reported by the volunteers. The current research demonstrates that this

unique herbal extract blend LI85008F, combined with modest calorie restriction and physical

activity, is effective for weight management in overweight men and women.

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 Conflict of Interest

KD and DVK are employees of Krupa Centre for Diabetes and Obesity and Sudeep Diabetes

Care Centre, Bengaluru, India. KVA is an employee of Laila Nutraceuticals, India. BAD is an

employee of PLT Health Solutions Inc, NJ, USA. This study was funded by Laila

Nutraceuticals, India and PLT (grant# C007185); KD and DVK were the grant recipients.
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Authorship

The authors’ responsibilities were as follows—AVKR and BAD: designed the research; KD and

DVK: conducted the research; Laila Nutraceuticals under the direction of AVKR: produced the

intervention products; KD, DVK, AVKR and BAD: analysis and interpretation of data; AVKR

and BAD: wrote the manuscript; BAD: had primary responsibility for the final content; and all

authors: read and approved the final manuscript.

Acknowledgements

The authors thank Sri. G. Ganga Raju, Chairman, Mr. G. Rama Raju, Director Laila Group and

Mr. B. Kiran CEO, Laila Nutraceuticals for encouragement and generous support. In addition,

the authors are thankful to Paul Flowerman, Chairman and Seth Flowerman, President of PLT

Health Solutions for their support.

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 Table 1: Demographic and baseline characteristics of LI85008F and Placebo groups.

LI85008F Placebo P value*

Characteristics
(n=70) (n =70)
Gender
Men (%) 26(44.8) 32(55.17) -
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Women (%) 44(53.66) 38(46.34) -

Age (yrs.) 35.16 ± 9.296 37.26 ± 9.756 0.194

Weight (Kg) 75.73 ± 9.710 75.11 ± 10.295 0.714
Height (cm) 162.03 ± 9.347 162.19 ± 9.555 0.920
BMI (Kg/m2) 28.71 ± 0.894 28.41 ± 1.048 0.070
Waist circumference
98.64 ± 9.928 97.86 ±7.980 0.609
(cm)
Hip circumference
107.41 ± 10.628 105.41 ± 8.511 0.221
(cm)
Waist: Hip ratio 0.92 ± 0.086 0.93 ± 0.076 0.468
Values represent mean±SD
* two tailed unpaired t test between LI85008F and Placebo

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 Table 2: Reductions in anthropometric variables in LI85008F and placebo groups from
the baseline through week 16

LI85008F Placebo
Parameters Week P-value#
(n=66) (n=64)
2 0.67±0.365 0.19±0.305 <0.0001
4 1.69±0.714 0.27±0.421 <0.0001
Body weight (kg) 8 2.77±0.957 0.48±0.609 <0.0001
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12 4.03±1.299 0.67±1.105 <0.0001

16 5.36±1.769 0.87±1.381 <0.0001
2 0.25±0.125 0.07±0.125 <0.0001
4 0.64±0.257 0.10±0.171 <0.0001
BMI (kg/m2) 8 1.06±0.359 0.19±0.245 <0.0001
12 1.54±0.503 0.26±0.450 <0.0001
16 2.05±0.693 0.34±0.559 <0.0001
2 1.32±0.729 0.22±0.851 <0.0001
4 2.52±0.938 0.43±0.930 <0.0001
Waist Circumference
8 3.44±1.181 0.78±1.302 <0.0001
(cm)
12 4.35±1.316 1.14±1.473 <0.0001
16 5.38±1.602 1.70±1.827 <0.0001
2 1.43±0.837 0.31±0.986 <0.0001
4 2.73±1.322 0.45±1.127 <0.0001
Hip Circumference
8 3.28±1.465 0.67±1.251 <0.0001
(cm)
12 3.82±1.590 0.86±1.492 <0.0001
16 4.49±1.664 1.19±1.465 <0.0001
2 0.00005±0.008 -0.00064±0.010 0.6805
4 0.0003±0.013 0.0003±0.012 0.9930
Waist/Hip Ratio 8 0.0043±0.014 0.0015±0.014 0.2574
12 0.00833±0.017 0.0034±0.015 0.0829
16 0.0123±0.020 0.0059±0.017 0.0547
Values represent mean±SD
# the comparisons between changes in placebo and treatment groups were analyzed using
unpaired t-test
Negative (-) values indicate increase in measures.

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 Table 3: Comparison of body composition estimated by DEXA

LI85008F Placebo
Variable P-value(a)
(N=65) (N=64)
Fat mass
Baseline (kg) 33.17 ± 9.567 30.60 ± 7.895 0.0986
Week 16 (kg) 32.06 ± 9.379 31.20 ± 8.202 0.5827
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Mean Change in baseline

-1.11 ± 7.059 0.61 ± 5.052 0.1151
Fat mass (kg)
P-value(b) 0.2098 0.3395
Lean mass
Baseline (kg) 41.26 ±7.773 41.37 ± 8.137 0.9382
Week 16 (kg) 42.16 ± 8.714 40.46 ± 7.453 0.2363
Mean Change in baseline
0.90 ± 3.743 -0.91 ± 3.621 0.0061*
lean mass (kg)
P-value(b) 0.0570 0.0490*
Values indicate mean ± SD. (-) values indicate loss or reduction of weight. P-value (a): Unpaired
t-test between the groups. P-value (b): Paired t-test between baseline and the end of intervention.
*indicates significance (P<0.05).

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 Table 4: Changes in serum fat metabolism markers in LI85008F and Placebo groups
from baseline
LI85008F Placebo
Parameters Week P-value#
(n=66) (n=64)
4 2.57±6.173 0.25±6.402 0.0375
HDL (mg/dL) 8 5.23±7.460 -2.02±6.903 <0.0001
16 8.66±8.908 -3.51±5.543 <0.0001
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4 -12.52±12.229 1.59±12.261 <0.0001

LDL (mg/dL) 8 -23.53±17.700 0.63±13.151 <0.0001
16 -30.20±18.387 -1.08±9.519 <0.0001
4 -2.80±4.558 -0.06±3.399 0.0002
VLDL (mg/dL) 8 -4.67±5.079 -0.78±3.722 <0.0001
16 -6.80±6.369 -1.09±3.059 <0.0001
4 -12.35±14.650 2.04±12.632 <0.0001
Total Cholesterol
8 -22.57±20.248 -1.81±14.957 <0.0001
(mg/dL)
16 -28.04±21.805 -5.37±10.203 <0.0001
4 -15.06±19.796 0.02±16.559 <0.0001
Triglycerides
8 -23.18±25.364 -3.17±18.306 <0.0001
(mg/dL)
16 -34.12±31.682 -5.09±14.990 <0.0001
4 -0.31±0.361 0.02±0.401 <0.0001
LDL/HDL Ratio 8 -0.59±0.423 0.09±0.399 <0.0001
16 -0.78±0.460 0.12±0.352 <0.0001
Values represent mean±SD
# the comparisons between changes in placebo and treatment groups were analyzed using
unpaired t-test Negative (-) and positive values indicate decrease and increase in measures,
respectively.
HDL, High Density Lipoprotein: LDL, Low Density Lipoprotein; VLDL, Very Low-Density
Lipoprotein.

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 Figure Legends

Figure 1: A typical High-Performance Liquid Chromatography (HPLC) chromatogram is

showing the major components of LI85008F. Total curcuminoids, Mahanine and Quercetin 3-O-

glycoside have been identified at 254 nm. The results are plotted in arbitrary units (AU) versus
Accepted Article

elution time (min).

Figure 2: CONSORT flow diagram of subject disposition. Eligible subjects (n=140) were

randomized into LI85008F or placebo groups. Final analysis was conducted on per protocol

population.

Figure 3: Scatter diagram presents reduction of the baseline body weight of the individual

participants in LI85008F supplemented and placebo groups at week 16.

Figure 4: LI85008F modulates Adiponectin and Ghrelin levels in Serum. In Panel A, each bar

represents the serum adiponectin concentration (mean±SD) of participants supplemented with

LI85008F and placebo at the baseline and at week 16, respectively. In Panel B, each bar

represents the serum Ghrelin concentration (mean±SD) of participants supplemented with

LI85008F and placebo at the baseline and at week 16, respectively. LI85008F (n=44) and

placebo (n=55). Paired t-test was performed for intragroup comparison between baseline and

week 16, * indicates significance at <0.05.

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Accepted Article Figure 1

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 CONSORT 2010 Flow Diagram
Accepted Article

Enrollment Assessed for eligibility (n= 157)

Excluded (n= 17)

♦ Not meeting inclusion criteria (n= 09)
♦ Declined to participate (n= 08)
♦ Other reasons (n= 00)

Randomized (n= 140)

Allocation
Allocated to Placebo (n= 70) Allocated to Treatment (n= 70)
♦ Received allocated intervention (n= 70) ♦ Received allocated intervention (n= 70)
♦ Did not receive allocated intervention ♦ Did not receive allocated intervention
(give reasons) (n= 00) (give reasons) (n= 00)

Follow-Up
Lost to follow-up (give reasons) (n= 00) Lost to follow-up (give reasons) (n= 00)

Discontinued intervention (Subject’s Discontinued intervention (Subject’s

decision to discontinue) (n= 06) decision to discontinue) (n= 04)

Analysis
Analysed (n=64) Analysed (n=66)
♦ Excluded from analysis (give reasons) (n= 0) ♦ Excluded from analysis (give reasons) (n= 0)

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 Figure 3
Accepted Article

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Accepted Article Figure 4

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