DESC:FIELD OF THE DISCLOSURE
The present disclosure relates to a herbal composition for the management of obesity. More particularly, the present disclosure relates to a dietary supplement for the management of obesity.

BACKGROUND
Obesity is a condition which may be attributed to the modern day lifestyle that encompasses sedentary work-culture, high fat, calorie-rich diet, dearth of regular exercise or physical activity, addiction to habit forming substances such as tobacco and alcohol and high day-to-day stress levels. Obesity has reached epidemic proportions globally, with more than 1 billion adults overweight - at least 300 million of them being clinically obese - and is a major contributor to the global burden of chronic disease and disability. Ischemic heart diseases often referred to as lifestyle diseases, have obesity as one of their root causes. Ischemic heart disease is among the major causes of death in the world today according to a recent World Health Organization (WHO) report (published at http://who.int/mediacentre/factsheets/fs310/en/). Cardiovascular diseases (CVDs) were responsible for causing the death of nearly 17 million people in the year 2011, which amounts to 3 in every 10 deaths. The importance of managing obesity is thus evident.

Often coexisting in developing countries with under-nutrition, obesity is a complex condition with serious social and psychological dimensions and affects virtually all ages and socio-economic groups. Obesity and overweight pose a major risk for other serious chronic diseases, including type-2 diabetes, hypertension, stroke and certain forms of cancer. The health consequences range from increased risk of premature death, to serious chronic conditions that reduce the overall quality of life.

It is therefore safe to state that managing obesity would substantially aid in reducing global mortality, increasing life expectancy and increasing the quality of life. Dietary changes, exercise and activity, behavioral change, prescription weight-loss medications and weight-loss surgery are the common treatment arms for managing obesity. The treatment method to be undertaken often depends on the preferred choice of an individual undergoing treatment as well as the level of obesity.

The most preferred treatment method for weight loss is dieting and physical exercise. However, due to busy schedules and sedentary lifestyles, following-up with these two methods, on a regular basis, becomes difficult. Weight loss surgery, on the other hand, is generally ruled out by most people due to the high costs involved. Therefore, there is a gradual shift towards the use of drugs.

The drugs used for weight-loss, generally alter one of the fundamental processes of the human body, such as altering the appetite, metabolism or absorption of calories. Orlistat is the only anti-obesity medication which is currently approved by the FDA for long term use. It reduces intestinal fat absorption by inhibiting the pancreatic enzyme lipase. Rimonabant and Sibutramine are the other drugs that had initially been approved for the treatment of obesity, but were banned eventually due to safety concerns. Because of the potential side effects, it has been recommended that anti-obesity drugs be only prescribed for obesity in cases where it is hoped that the benefits of the treatment outweigh its risk.

In view of the above, nutrition-based interventions are being hailed as an inexpensive alternative to aid weight loss and to manage weight in a better way. Medicinal herbal supplements, being cost-effective and exerting less to no toxic side-effects, are being extensively utilized due to their effectiveness in managing many chronic disorders.

Various attempts have been made by the scientific community in managing obesity. Some representative patent documents that disclose the use of formulations for controlling obesity are discussed herein below.

United States Patent Application 20060159773 discloses herbal compositions containing Hoodia gordonii and synergistically enhancing ingredients such as green coffee bean extract for controlling obesity and for supporting the treatment of various health conditions related thereto.

WIPO Patent Application WO/2012/140666 discloses extracts of Cassia auriculata (CA), compositions comprising an extract of the same and pharmaceutically active compounds isolated from Cassia auriculata which are useful for the treatment of various disorders including obesity.

Various herbal formulations have also been disclosed, however, these formulations are associated with several drawbacks such as they do not provide the desired weight control along with other health benefits. Therefore, there is a need to provide an improved composition for weight control and other preferred health benefits.

The inventors of the present disclosure therefore envisage a cost-effective and safe herbal composition or a dietary supplement which can be used for the management of obesity.

OBJECTS
Some of the objects of the present disclosure, which at least in one embodiment is adapted to provide, are described herein below:
It is an object of the present disclosure to develop a process for the preparation of an extract of Mangifera indica with enhanced proportion of flavonoids and polyphenols.
It is another object of the present disclosure to provide a herbal composition for management of obesity.
It is another object of the present disclosure to provide a herbal composition for management of obesity, which is cost-effective and non-toxic.
It is yet another object of the present disclosure to provide a herbal composition for improving high density lipoprotein (HDL) levels.
It is still another object of the present disclosure to provide a process for the preparation of a herbal composition.
Other objects and advantages of the present disclosure will be more apparent from the following description which is not intended to limit the scope of the present disclosure.

SUMMARY OF THE DISCLOSURE:
In one aspect of the present disclosure there is provided a process for the preparation of a Mangifera indica extract; the process comprising of following steps:
i. obtaining plant material from Mangifera indica;
ii. treating the plant material by at least one operation selected from the group consisting of sorting, selecting, cleaning, washing, rinsing, drying, grinding, extracting and pulverizing to obtain a treated plant material; and
iii. obtaining an extract of Mangifera indica comprising predetermined quantities of specific parts of Mangifera indica by an iterative extraction method using at least one solvent followed by concentrating and drying the extract.
In accordance with another aspect of the present disclosure there is provided I extract, obtained by the process of the present disclosure, wherein the extract is enriched with polyphenolic compounds comprising gallic acid, 3, 4-dihydroxy benzoic acid, methyl gallate, proplyl gallate, pyrogallol, parahydroxy benzoic acid, vanillic acid, syringic acid, ferulic acid and ethyl gallate; and flavonoids comprising mangiferin, catechin, epicatechin and quercetin dehydrate.

In accordance with yet another aspect of the present disclosure there is provided an herbal composition for managing obesity comprising 60% to 90% powdered extract of dried bark, 5% to 20% powdered extract of dried leaves and 5% to 20% of powdered extract of dried skin of ripe fruit said having moisture between 1% to 3%.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
Figure 1 illustrates HPLC chromatogram of the herbal composition of the present disclosure showing mangiferin peak.
Figures 2(a)-2(f) illustrate HPLC chromatograms of the herbal composition of the present disclosure showing polyphenolic and flavonoidal constituents.
Figure 3(a) illustrates in-vitro evaluation of H2O2 scavenging activity of the herbal composition of the present disclosure.
Figure 3(b) illustrates in-vitro evaluation of SOD activity of the herbal composition of the present disclosure.
Figure 3(c) illustrates in-vitro evaluation of anti-oxidant activity of the herbal composition of the present disclosure.
Figure 3(d) illustrates in-vitro evaluation of anti-lipase activity of the herbal composition of the present disclosure.
Figure 3(e) illustrates in-vitro evaluation of carbonic anhydrase activity of the herbal composition of the present disclosure.
Figure 3(f) illustrates in-vitro evaluation of lipid peroxidation inhibitory activity of the herbal composition of the present disclosure.

DETAILED DESCRIPTION
Mangifera indica: Nomenclature
Botanical Species:
Mangifera indica L. Family: Anacardiaceae
Common Names:
Ambrah, Madhuulii, Madhuula, Madhuulaka (Sanskrit); Mango (English); Aam (Hindi); Ambiram, Mambazham, Mambalam, Mangai (Tamil); Amb (Punjabi); Ambo, Keri, Marvo (unripe) (Gujarati); Amb (Kashmiri); Amram, Choothaphalam, Manga, Manpalam, Mavu, (Malayalam); Amchur, Amba (Marathi).
Distribution:
Mangifera indica is a large evergreen tree belonging to Anacardiaceae family which is native to tropical Asia and has been cultivated in the Indian subcontinent for over 4000 years. Mango is native to southern Asia, especially eastern India, Burma, and the Andaman Islands.

Cultivation, Harvesting, and Post harvesting techniques:
Adequately drained and mildly acidic soil with pH 6-7 is preferred. Seasonally wet/dry climate zones of the lowland tropics, or frost-free subtropical areas are preferred. The plant ceases growth at temperatures below 55-60°F. Leaves and fruit are injured by mild frost (28-32°F).
Botanical Description:
Mangifera indica is a large evergreen tree in the Anacardiaceae family that grows to a height of 10-45 m, dome shaped with dense foliage, typically heavy branched from a stout trunk. The leaves are linear-oblong, pointed at both ends and spirally arranged on branches. The inflorescence occurs in panicles consisting of about 3000 tiny whitish-red or yellowish-green flowers. The fruit is a well known large drupe, but shows a great variation in shape and size. It contains a thick yellow pulp, single seed and thick yellowish-red skin when ripe. The seed is solitary, ovoid or oblong, encased in a hard, compressed fibrous endocarp.

In a first aspect, the present invention envisages a process for the preparation of a Mangifera indica extract, the process comprising the following steps.

In a first step, plant material is obtained from Mangifera indica. The plant material includes but is not limited to leaves, flowers, seeds, stem, branches, bark, roots, skin of ripe fruit and combinations thereof. The extraction process may employ any part of the Mangifera indica as a raw material either in wet or dry form.
In one embodiment of the present disclosure bark, leaves and ripe fruit skin are used for preparing the extract of Mangifera indica with enhanced phytochemical yield.

In the next step, the selected plant material is treated to obtain a treated plant material meant for extraction. The process involves pre-treatment of the plant material which includes but is not limited to sorting, cleaning, washing, rinsing, drying, grinding, extracting and pulverizing at least one part of the plant.

In the last step, an extract of Mangifera indica is obtained from the treated plant material by an iterative extraction method using at least one solvent followed by concentrating and drying the extract.
In accordance with a preferred embodiment of the present disclosure, a combination contains of three parts of Mangifera indica plant; namely the bark 60% to 90%, the dried leaf 5% to 20% and the dried skin of ripe fruit 5 % to 20%
The process for extraction envisaged in accordance with the present disclosure for preparation of an extract employs a series of extraction techniques carried out in any sequence which are selected from the group which includes but is not limited to hot stirring, reflux conventional extraction method, percolation, decoction, supercritical fluid extraction and the like.
The extract is selected from the group that includes but is not limited to alcoholic, hydro-alcoholic and aqueous extract.
The solvent used for extraction is a polar organic solvent and/ or a non-polar organic solvent.
In one embodiment of the present disclosure, the solvent includes but is not limited to water, methanol, ethanol, butanol, hexane, acetone, chloroform, petroleum ether and acetonitrile.
In one embodiment of the present disclosure, the solvent is ethanol.
In accordance with the embodiment of the present disclosure, the Mangifera indica extract is prepared by using 60 % to 80% v/v ethanol.
In accordance with the present disclosure the extraction is carried out at a temperature ranging from 70°C to 80°C for a time period of 5 to 10 hours.
The extract is dried at a temperature ranging from 80°C to 85°C to obtain a dried extract.

In accordance with another aspect of the present disclosure there is provided Mangifera indica extract, which is enriched with polyphenols and flavonoids. The polyphenols present in the extract include but are not limited to gallic acid, 3, 4-dihydroxy benzoic acid, methyl gallate, proplyl gallate, pyrogallol, parahydroxy benzoic acid, vanillic acid, syringic acid, ferulic acid and ethyl gallate. The flavonoids present in the extract include but are not limited to mangiferin, catechin, epicatechin and quercetin dehydrate.

The Mangifera indica extract comprises flavonoids and polyphenols.
The flavonoids present in the herbal composition include but are not limited to mangiferin, catechin, epicatechin and quercetin dehydrate.
The amount of flavonoids ranges from 27.63% to 29.49% of the total mass of the herbal composition.
The polyphenols present in the herbal composition include but are not limited to gallic acid, 3, 4-dihydroxy benzoic acid, methyl gallate, proplyl gallate, pyrogallol, parahydroxy benzoic acid, vanillic acid, syringic acid, ferulic acid and ethyl gallate.
The amount of polyphenols ranges from 17.1% to 20.7% of the total mass of the herbal composition.

In one embodiment of the present disclosure, the herbal composition comprises Mangifera indica extract obtained from a combination of three parts of the Mangifera indica plant; namely the bark, the dried leaf and the dried ripe fruit skin.

The herbal composition of the present disclosure may be given in the form of a daily adult dosage. Table 1 provides daily adult dose to be administered to a subject.

Table 1: Daily adult dosage of the herbal composition of the present disclosure:

Sr. No. Herbal composition Daily Adult Dose (mg)
1 Herbal composition 2250

In one embodiment of the present disclosure, the daily adult dose presented in Table 1 may be divided into multiple doses per day administered at time intervals throughout a day.
In one embodiment of the present disclosure, the daily adult dose is divided for administration as one, two, three, four, five, or six doses per day.
In one embodiment of the present disclosure, the daily dose is orally administered in three doses of herbal composition, to be administered 30 minutes before every meal thrice a day.
In one embodiment of the present disclosure, the composition comprises Mangiferin, flavonoids other than Mangiferin, and polyphenols other than Mangiferin.

In another embodiment of the present disclosure, the composition comprises 21.5±0.82% w/w of Mangiferin, 6.94±0.62% w/w of flavonoids other than Mangiferin and 18.9±1.8% w/w of polyphenols other than Mangiferin.

In accordance with yet another aspect of the present disclosure there is provided a use of the herbal composition of the present disclosure which includes but is not limited to preventing oxidation (antioxidant activity), controlling and/ or reducing body weight and/ or obesity, management of hyperlipidemic conditions, reducing oxidative stress, maintaining lipid profile and regulating the blood glucose levels. Significantly, the composition is non-toxic and non-mutagenic. The herbal composition of the present disclosure achieves reduction in weight by inhibiting fat absorption, decreasing fat synthesis and enhancing metabolism.

The present disclosure is further described in the light of the following non-limiting examples which are set forth for illustration purpose only and not to be construed for limiting the scope of the disclosure.

Example 1: Process for the preparation of the herbal composition of the present disclosure (HC)
Dried parts of Mangifera indica bark, leaf and fruit skin (80:10:10 ratio) were cut into small pieces, approximately 10 mm in length, to form a 100 kg mass. This mass was pulverized to form a coarse powder in a hammer mill and stacked in a vertical 1.0 KL extractor. The lower end of the extractor contained a perforated plate on which a filtration cloth was fixed. The bottom of the extractor was connected to a transfer pump, input and the output of the transfer pump was connected to a T-bend. One end was connected to the extractor top for circulation of the extraction mass during the extraction period and the other end of the T-bend was connected to a receiver tank.
The above-mentioned coarse powder was initially extracted with six bed volumes of 70% v/v ethyl alcohol (aqueous).The extraction was carried out at 75-78°C for about 7-8 hours with continuous circulation of the extract with a transfer pump. After completion of extraction, the extract was filtered through a 5 micron SS candle filter and the resultant clear extract was collected in a receiver tank. The bed was re-extracted by adding 4 bed volumes of 70% ethyl alcohol 3 more times at a temperature of 75-78°C for about 7-8 hours. All the extracts were then collected in a receiver tank and the combined extract was concentrated in a reactor under vacuum at 80-85°C till the extract mass- total dissolved solids (TDS) reached 60-70%. The extract mass was further dried in a vacuum tray drier at 80-85°C till the completion of drying. The extract was then mixed with a preservative, to yield the herbal composition of the present disclosure. The yield of the herbal composition of the present disclosure was found to be 14±0.5 w/w%.

Phyto-chemical analysis:
The composition was further subjected to phyto-chemical analysis through Spectrophotometric and HPLC estimations. The studies confirmed that the herbal composition of the present disclosure is associated with a good percentage of antioxidant constituents.

Spectrophotometric Analysis:
The herbal composition of the present disclosure was subjected to spectroscopic analysis. The results are provided in Table No. 2
Table 2: Results of phyto-chemical analysis of the herbal composition of the present disclosure

Sr. No. Phyto-constituents Percentage Analysis Method
1 Total Flavonoids 28.56±0.93w/w% Spectrophotometric
2 Total polyphenols 18.9±1.8 w/w% Spectrophotometric

Conclusion:
The results revealed that the active ingredients in the full spectrum of the herbal composition of the present disclosure are polyphenols and flavonoids. The composition provided herein utilize a unique full spectrum of herbal composition containing greater than 28% polyphenols and greater than 18% flavonoids as analyzed by Spectrophotometric method.

High performance liquid chromatography (HPLC) analysis:
The composition of the total flavonoids was determined by the HPLC method. The results are provided in Table No. 3.

Table 3: Composition of the determined flavonoids in the herbal composition of the present disclosure
Active Ingredients Per 100g (Flavonoids)
S.No. Name of the active ingredient Approx. w/w % of active ingredient
1 Mangiferin 21.5±0.82
2 (+)- Catechin 3.84±0.31
3 (-)- Epicatechin 2.67±0.25
4 Quercetin dihydrate 0.43±0.06

The composition of the total polyphenols was determined by the HPLC method. The results are provided in Table No. 4.

Table 4: Composition of the determined polyphenols in the herbal composition of the present disclosure

Active Ingredients Per 100g (Polyphenols)
S.No. Name of the active ingredient Approx. w/w % of active ingredient
1 Gallic acid 4.5±0.42
2 3, 4-Dihydroxy benzoic acid 0.93±0.23
3 Methyl gallate 1.2±0.34
4 Proplyl gallate 2.31±0.73
5 Pyrogallol 0.48±0.08
6 Parahydroxy benzoic acid 1.43±0.07
7 Vanillic acid 0.33±0.06
8 Syringic acid 0.82±0.13
9 Ferulic acid 0.16±0.02
10 Ethyl gallate 0.76±0.14

Conclusion:
The results of high performance liquid chromatography (HPLC) revealed that the herbal composition of the present disclosure containing 750 mg of Mangifera indica extract has greater than 150 mg mangiferin, an anti-obesity agent along with other constituents (Figure-1).
Further, the results revealed that the herbal composition of the present disclosure comprises of polyphenols and flavonoids. (Figure-2(a)-(f))

Example 2: In-vitro studies of the herbal composition (HC) of the present disclosure:
A]: In-vitro Antioxidant activity studies:
i) Hydrogen peroxide (H2O2) scavenging activity:
Procedure:
Hydrogen peroxide scavenging activity of the HC was estimated in this study where initially, HC solutions were prepared at different concentrations in distilled water and the resultant solutions were mixed in 0.6 ml of 40 mM H2O2 solution prepared in phosphate buffer (0.1 M pH 7.4) and incubated for 10 min. The absorbance was measured at 230 nm against a blank solution containing H2O2, without the HC (Control). Similar procedure was repeated for Mangiferin and Quercetin and the amount of hydrogen peroxide radical inhibited was calculated using the following equation.
The result is expressed as % H2O2 radical scavenging activity in Table No. 5

H2O2 radical scavenging activity = (Abs control – Abs sample) × 100
(Abs control)

where:
Abs control - Absorbance of H2O2 radical
Abs sample - Absorbance of sample extract/ standard (the HC, mangiferin and quercetin)

Table 5: H2O2scavenging activity
H2O2scavenging activity of the herbal composition (HC) of the present disclosure
Samples Concentration in µg/ml
10 20 30 40
Quercetin(Standard) 5.6±0.10 14.55±0.17 18.86±0.15 33.96±0.14
Mangiferin 98% 8.49±0.24 33.01±0.34 64.15±0.45 76.41±0.19
HC 16.43±0.55 19.17±0.08 53.42±0.32 75.34±0.42

Conclusion: The HC exhibited good H2O2 scavenging activity in concentration dependent manner. (Figure-3(a))

ii) Superoxide Dismutase (SOD) Activity:
Procedure:
The superoxide anion scavenging activity of the HC was measured in this test. 1 ml (156 µM) of nitro blue tetrazolium (NBT), 1 ml (468 µM) of nicotinamide adenine dinucleotide (NADH) and 3 ml of the HC at 100 µg/ml concentration were admixed in phosphate buffer (pH 7.4). The reaction was started by adding 100 µl of phenazine methosulfate (PMS) (0.18mg/ml) and the mixture was incubated at 25°C for 5 min followed by measuring the absorbance at 560 nm. Similar solutions were prepared for quercetin and mangiferin and the percentage of inhibition was calculated by using the formula provided herein-below; the result is expressed as % superoxide dismutase scavenging activity in Table No. 6

% superoxide dismutase inhibition = (Abs control – Abs sample) × 100
Abs control
where:
Abs control - Absorbance of control
Abs sample - Absorbance of sample extract/ standard (the HC, mangiferin and quercetin)

Table 6: SOD activity results
SOD activity
Sample Concentration in µg/ml % Inhibition
Quercetin (Standard) 51.03±0.44
Mangiferin 98% 7.50±0.14
HC 83.40±0.36

Conclusion: HC exhibited significant SOD activity in comparison with 98% Mangiferin and quercetin (Figure-3(b)).

iii) Total antioxidant assay:
Procedure:
The total antioxidant activity of the HC was determined by the phosphomolybdenum method where 0.3 ml of the HC (100µg/ml) was combined with 3ml of a reagent solution (0.6M sulfuric acid, 28mM sodium phosphate and 4mM ammonium molybdate). The resultant reaction mixture was incubated at 95°C for 90 min and cooled to room temperature. The absorbance of the solution was measured at 695 nm against a blank solution (water). The total antioxidant capacity is expressed as the milligram equivalents of ascorbic acid in Table No. 7
Table 7: Total antioxidant assay
Total antioxidant assay
Sample Concentration µg /ml mg equivalent ascorbic acid
Mangiferin 98% 31.23 ±0.34
HC 41.06 ±0.20

Conclusion: The total antioxidant capability of the HC was found to be 41.06 mg equivalent ascorbic acid (Figure-3(c)).
The HC exhibited good antioxidant property. The antioxidant ability of HC was confirmed through hydrogen peroxide radical scavenging activity, superoxide radical scavenging activity and the total antioxidant capacity.

B] In vitro anti-obesity studies:
i) Anti-lipase assay:
Procedure:
A suspension containing 1% (V/V) of triolein and 1% (V/V) of tween 40 in 0.1M phosphate buffer (pH-8) was prepared and emulsified. The assay was then initiated by adding 800µl of the Triolein emulsion to 200µl of Porcine Pancreatic Lipase (0.5 g Pancreatic Lipase in 15 ml of 0.1 M phosphate buffer at pH-8) and 200 µl of the HC extract (100 µg/ml concentration). The resultant mixture was admixed and the absorbance was measured immediately at 450 nm (T0). The mixtures were then incubated at 37 oC for 30 min and the absorption was measured at 450 nm (T30). The results expressed as % inhibition are provided in Table No. 8

Inhibition % = A(cont)- A(test) ×100
A(cont)

where:
A(cont) - is the absorbance of the control (TC0-TC30)
A(test) - is the absorbance of the sample test /standard (T0-T30) (the HC, mangiferin and quercetin)

Table 8: Anti-lipase assay results
Anti-lipase assay
Sample Concentration: 1.2 mg/ml % Inhibition
Orlistat 75.27±0.35
Mangiferin 98% 41.78±0.40
HC 42.30±0.27

Conclusion: The HC showed good anti-lipase property of more than 42% (Figure-3(d)).

ii) Carbonic Anhydrase Assay:
Procedure:
The HC at 250 µg/ml and 98% Mangiferin were independently admixed in 1.4 ml of tris buffer and 2.5 ml of carbonic anhydrase. The resultant mixtures were incubated at room temperature for 5 min. After incubation, 1 ml of 4-NPA was added to the mixtures and further incubated for 15-20 min at 37 oC. The absorbance of the reaction mixtures were measured at 400 nm. The results expressed as % inhibition are provided in Table No. 9

Inhibition % = A(cont)- A(test) ×100
A(cont)

where:
A (cont) - is the absorbance of the control
A (test) - is the absorbance of the test sample /standard

Table 9: Carbonic Anhydrase assay results
Carbonic Anhydrase
Sample Concentration: 100µg % inhibition
Mangiferin 98% 47.06±0.56
HC 52.31±0.70

Conclusion: The HC showed enhanced anti-lipase property as compared to Mangiferin (Figure-3(e)).
(iii) Lipid-peroxidase inhibitory activity:
Procedure: Take 0.5ml of egg homogenate (10%in distilled water) and 0.1 ml of HC in the concentration range 100-500µ g/ml in a test tube and volume made up to 1 ml by adding distilled water. Finally add 0.05 ml ferrous sulphate (0.07M) to the above mixture and incubate for 30 min to induce the lipid peroxidation. Thereafter add 1.5 ml of 20% acetic acid (pH 3.5) and 1.5 ml of (0.8%in 1.1% SDS) TBA and 0.05 ml (20%) TCA then heated in boiling water bath for 60 min. After cooling 5 ml of butanol will be added to all the test tubes and centrifuged at 3000 rpm for 10 min. Measure the absorbance of the organic upper layer at 532 nm. The percentage inhibition of lipid peroxidation calculated from the formula,

The results expressed as % inhibition are provided in Table No. 10

Inhibition % = ( Abcontrol – Abtest sample ) *100
Abcontrol

where:

Table 10: Lipid-peroxidase inhibitory activity results
Lipid-peroxidase inhibitory activity
Sample Concentration: % Inhibition
Quercetin 53.44±0.19
Mangiferin 98% 74.9±0.56
HC 56.52±0.25
Conclusion: The Herbal composition shows better prevention towards generation of lipid peroxides with percent inhibition of 56.52 %. (Figure-3(f))

Example 2: In-vivo studies of the herbal composition of the present disclosure:
A] Acute oral toxicity study in rats with the HC
Single-dose oral toxicity of the HC was evaluated in albino Wistar rats. A limit test was performed in which female rats received a single oral administration of the HC at a dose of 2000 mg/kg body weight (b.w.). Following dosing, the limit test rats were observed daily and weighed weekly. A gross necropsy examination was performed on all limit test animals at the time of scheduled euthanasia (day 14).No mortality occurred during the duration of the limit test. Further, no significant gross internal findings were observed at necropsy on study day 14.
Conclusion: Under the conditions of this test, the acute oral lethal dose (LD50) of the HC was estimated to be greater than 2000 mg/kg in rats.

B] Repeated Dose 28 Day Oral Toxicity Study of the HC
Repeated dose 28 day oral toxicity study, with the HC in rats followed by 14 day recovery period, was performed. The objective of the study was to assess the safety of the HC when administered orally to female and male rats. Another objective was to determine the target organ toxicity, no observed effect level (NOEL) and reversibility of signs of toxicity after the recovery period.
Wistar female and male rats were treated with the HC 250, 500 and 1000 mg/kg/b.w for 28 days by oral gavage, followed by 14 day recovery period.

Observations:
1) All the male and female animals from control and all the treated dose groups up to 1000 mg/kg survived throughout the dosing period of 28 days and the recovery period of 14 days.
2) No signs of intoxication were observed in male and female animals from different dose groups during the dosing period of 28 days and during the recovery period of 14 days.
3) Male rats showed a significant decrease in body weight gain with 250 and 500 mg/kg b.w. of the HC and a significant decrease in body weight gain were seen with 500 and 1000 mg/kg b.w. of the HC in females when compared with the control on day 29. During the reversal period the animals restored to normal when compared with control reversal group on day 43.
4) Food consumption of control and treated animals was found to be comparable throughout the dosing period of 28 days and the recovery period of 14 days.
5) Hematological analysis conducted at the end of the dosing period on day 29 and at the end of recovery period on day 43, revealed no abnormalities attributable to the treatment.
6) Biochemical analysis conducted at the end of the dosing period on day 29 and at the end of recovery period on day 43, revealed no attributable changes to the treatment. Except there was a significant increase in high density lipoproteins (HDL) levels in female rats treated with 250, 500 and 1000 mg/kg/b.w. of the HC.
7) Functional battery observation tests conducted at termination revealed no abnormalities.
8) Urine analysis, conducted at the end of the dosing period in week 4 and at the end of recovery period in week 6 revealed no abnormality attributable to the treatment.
9) Organ weight data of male and female sacrificed at the end of the dosing period and at the end of the recovery period was found to be comparable with that of respective controls.
10) Gross pathological examination did not reveal any abnormality.
11) Histopathological examination did not reveal any major abnormality except congestion of blood vessels in few organs.
Conclusion: Based on the above findings, no observed adverse effect level (NOAEL) of the HC was found at the dose of 1000 mg/kg/b.w. for both female and male Wistar rats when given orally for 28 days followed by a 14 day recovery period.

C] Repeated Dose 90 Day Oral Toxicity Study of the HC
Repeated dose 90 day oral toxicity study, with the HC in rats followed by 14 day recovery period, was performed. The objective of the study was to assess the safety of the HC when administered orally to female and male rats. Another objective was to determine the target organ toxicity, no observed effect level (NOEL) and reversibility of signs of toxicity after the recovery period.
Albino wistar female and male rats were treated with 250, 500 and 1000 mg/kg/b.w. of the herbal composition of the present disclosure for 90 days by oral gavage, followed by 14 day recovery period.
Further, Genotoxicity test was performed to find out its mutagenic effect in mice by chromosomal aberration test and micronucleus test. The doses administered were 250, 500, 1000 and 2000 mg/kg orally to male and female albino mice for Genotoxicity using chromosomal aberration test and micronucleus test.

Observations:
All the male and female animals from control and all the treated dose groups up to 1000 mg/kg survived throughout the dosing period of 90 days and the recovery period of 14 days.
Conclusion:
This shows that the herbal composition of the present disclosure is safe and non toxic even if it is administered for the extended period of time at the dose of 1000mg/kg.
Genotoxicity studies result reveals that herbal composition is non mutagenic and safe at the dose of 2000mg/kg.

D] Anti-obesity activity of the HC with respect to Standard:
Objective: The objective was to investigate the effect of the HC against high fat diet fed rats.
Procedure:
Animals: Male Wistar rats (160 to 200 g)
No of animals: 36
Housing: 3/ cage, 12 hrs light/dark cycle
Temperature: 25±20c
Model & Composition of the Diet:
High fat diet in rats
? 25% Lard
? 5% Soyabean oil
? 5% starch
? 65% Normal commercial available rat feed
Groups:
Male Wistar rats were divided into six groups with six animals in each group.
Group 01: Normal rat feed + Vehicle (distilled water)
Group 02: High fat diet + Vehicle (distilled water)
Group 03: High fat diet + Standard drug (Orlistat 30 mg/kg)
Group 04: High fat diet + HC 1(50 mg/kg)
Group 05: High fat diet + HC 2 (100 mg/kg)
Group 06: High fat diet + HC 3 (150 mg/kg)
All the groups were fed with high fat diet for 42 days except one group which was considered as negative control group. All the data were analyzed by one way ANOVA followed by Dunnett’s t test. The results are provided in Table No. 11, Table No. 12, Table No. 13, Table No. 14, Table No. 15, Table No. 16, Table No. 17, and Table No. 18

Observations:

i) Effect of the HC on body weight gain in male rats

Table 11: Results of the effect of the HC on body weight gain in male rats
Groups Treatment
(42 days) Final weight Initial weight Difference in body weight gain
Group I Normal rat feed + Vehicle (distilled water) 251.70±2.02 181.70±1.99 70.00±2.60
Group II High fat diet + Vehicle (distilled water)
292.50±1.81 181.50±1.31 111.0±2.20*** a
Group III High fat diet + Standard drug (Orlistat 30 mg/kg)
261.80±2.53 182.00±0.96 79.82±2.58*** b
Group IV High fat diet + HC 1(50 mg/kg)
254.80±1.87 182.30±1.99 72.43±3.16***b
Group V High fat diet + HC 2 (100 mg/kg)
250.50±2.53 183.30±0.88 67.15±2.75***b
Group VI High fat diet + HC 3 (150 mg/kg) 259.20±6.06 184.70±0.80 74.50±5.77*** b

Values are expressed in terms of SEM ±Mean.
Number of animals in each group n=6.
aComparison made with control group (Group I).
b Comparison made with high fat diet group (Group II).
***P<0.001.

Conclusion: The HC at all doses showed significant reduction (P<0.001) in body weight gain when compared to the high fat diet group.

ii) Effect of the HC on the average food intake in male rats

Table 12: Results of the effect of the HC on the average food intake in male rats
Groups Average food intake g/rat
Group I 17.21± 0.38
Group II 13.56±0.36***a
Group III 13.45±0.28nsb
Group IV 12.13±0.24**b
Group V 11.26±0.24***b
Group VI 13.18±0.31nsb

Values are expressed in terms of SEM ±Mean.
Number of animals in each group n=6.
aComparison made with control group (Group I).
b Comparison made with high fat diet group (Group II).
***P<0.001 ***P<0.001 **P<0.01 ns non significant.

Conclusion:
The HC at 50 and 100mg/kg b.w showed significant reduction (P<0.01, P<0.001) in food intake when compared to the high fat diet group.

iii) Effect of the HC on liver organ weight and mesenteric, brown adipose tissue (BAT), perirenal fat pads and epididymal fat pad

Table 13: Results of the effect of the HC on liver organ weight and mesenteric, brown adipose tissue (BAT), perirenal fat pads and epididymal fat pad.

Groups
Liver Mesenteric fat BAT Perirenal fat pad tissue Epididymal fat pad
Right Left Right Left
Group I
2.8±
0.01 0.59±
0.02 0.13± 0.01 0.25±
0.02 0.21±
0.01 0.38±
0.01 0.33±
0.02
Group II
3.5± 0.15***a 1.14± 0.07***a 0.30± 0.01***a 1.05± 0.13***a 1.07± 0.11***a 0.63±
0.04***a 0.56±
0.05***a
Group III
2.8± 0.08***b 1.02± 0.07nsb 0.28± 0.01nsb 0.77± 0.08*b 0.77±
0.08*b 0.55±
0.02nsb 0.52±
0.03nsb
Group IV
2.7± 0.07***b 0.80± 0.03**b 0.17± 0.01***b 0.49± 0.01***b 0.44± 0.03***b 0.51±
0.01*b 0.42±
0.01*b
Group V
2.8± 0.03***b 0.83± 0.03**b 0.17± 0.01***b 0.47± 0.02***b 0.67± 0.02*** b 0.51±
0.02*b 0.39±
0.02**b
Group VI
2.9± 0.02***b 0.84± 0.03**b 0.17± 0.01***b 0.63± 0.07**b 0.69± 0.04**b 0.51±
0.02*b 0.42±
0.03*b

Values are expressed in terms of SEM ±Mean.
Number of animals in each group n=6.
aComparison made with control group (Group I).
b Comparison made with high fat diet group (Group II).
***P<0.001 **P<0.01 * P<0.05 ns non significant.

Conclusion: The HC at all doses showed significant reduction (P<0.001) in liver organ weight, Perirenal fat pads and BAT, Mesentric fat (P<0.001), epididymal fat pad (P<0.05) when compared to the high fat diet group.

iv) Effect of the HC on serum glucose, total cholesterol, triglyceride, HDL-c and LDL-c levels (mg/dL)

Table 14: Results of the effect of the HC on serum glucose, total cholesterol, triglyceride, HDL-c and LDL-c levels (mg/dL)
Groups
Glucose
Total cholesterol
Triglyceride
HDL-c
LDL-c
Group I
81.63±2.96¬ 47.53±1.15 52.61±2.14 22.61±0.56 14.40±1.51
Group II
114.20±7.63***a 61.39±2.47***a 104.6±4.16***a 17.27±0.51***a 23.20±2.96*a
Group III
92.00±2.89**b 50.91±0.62***b 79.96±1.13***b 21.72±1.22**b 13.20±1.71**b
Group IV
96.23±2.72*b 48.96±1.90***b 71.48±5.42***b 23.77±0.99***b 10.89±1.02***b
Group V
82.37±2.82***b 48.93±2.23***b 55.92±2.03***b 24.97±0.87***b 12.77±1.75***b
Group VI
96.70±0.96*b 52.55±1.42**b 79.77±5.30***b 24.25±0.48***b 12.35±2.13**b
Values are expressed in terms of SEM ±Mean.
Number of animals in each group n=6.
aComparison made with control group (Group I).
b Comparison made with high fat diet group (Group II).
ns= not significant
***P<0.001 **P<0.01 * P<0.05

Conclusion:
The HC at 50 and 150 mg/kg b.w. showed a significant reduction (P<0.05) and at 100mg/kg b.w. showed significant reduction (P<0.01) in glucose levels when compared to high fat diet group.
The HC at 50 and 100 mg/kg b.w. showed a significant reduction (P<0.001) and at 150mg/kg b.w. showed significant reduction (P<0.01) in total cholesterol and low density lipoproteins (LDL) levels when compared to high fat diet group.
The HC at all doses showed a significant reduction (P<0.001) in total triglycerides and an increase in the HDL levels when compared to the high fat diet group (Group II).

v) Effect of the HC on atherogenic index
Table 15: Results of the effect of the HC on atherogenic index
Group Atherogenic Index
Group I 1.11±0.08
Group II 2.57±0.18***a
Group III 1.39±0.15***b
Group IV 1.07±0.06***b
Group V 0.96±0.06***b
Group VI 1.17±0.08***b
Values are expressed in terms of SEM ±Mean.
Number of animals in each group n=6.
aComparison made with control group (Group I)..
b Comparison made with high fat diet group (Group II)..
***P<0.001
Conclusion: The HC at all doses showed a significant reduction (P<0.001) in atherogenic index when compared to the high fat diet group.

vi) Effect of the HC on liver triglyceride levels

Table 16: Results of the effect of the HC on liver triglyceride levels
Group liver triglycerides
Group I 63.85±0.42
Group II 96.47±1.67***a
Group III 87.77±2.28*b
Group IV 78.57±1.93***b
Group V 86.47±2.11**b
Group VI 85.46±2.31**b
Values are expressed in terms of SEM ±Mean.
Number of animals in each group n=6.
aComparison made with control group (Group I).
b Comparison made with high fat diet group (Group II)..
***P<0.001**P<0.01 * P<0.05

Conclusion: The HC at 50 mg/kg b.w. showed a significant reduction (P<0.001) and at 100 & 150mg/kg b.w. showed significant reduction (P<0.01) in liver triglycerides levels when compared to the high fat diet group.

(vii) Effect of the HC on insulin hormonal levels

Table 17: Results of the effect of the HC on insulin hormonal levels
Group Insulin
(mU/L)
Group I 0.27±0.023
Group II 0.15±0.008***a
Group III 0.15±0.005nsb
Group IV 0.14±0.005nsb
Group V 0.22±0.016*b
Group VI 0.23±0.021**b
Values are expressed in terms of SEM ±Mean.
Number of animals in each group n=6.
aComparison made with control group (Group I).
b Comparison made with high fat diet group (Group II).
***P<0.001 **P<0.01* P<0.05 ns non significant.

Conclusion: The HC at 100 & 150mg/kg b.w. showed significant increase (P<0.05, P<0.01 respectively) in insulin levels when compared to the high fat diet group (Group II).

(viii) Effect of the HC on the fecal fatty acid estimation

Table 18: Effect of the HC on the fecal fatty acid estimation
Group Fecal fatty acid estimation
Group I 1.0±0.05
Group II 3.3±0.13***a
Group III 4.1±0.26*b
Group IV 2.3±0.13**b
Group V 2.4±0.26**b
Group VI 3.1±0.26nsb
Values are expressed in terms of SEM ±Mean.
Number of animals in each group n=6.
aComparison made with control group (Group I).
b Comparison made with high fat diet group (Group II).
***P<0.001 **P<0.01* P<0.05 ns non significant.

Conclusion: The HC at 50 & 100mg/kg b.w. showed significant reduction (P<0.01) in fecal fatty acids when compared to the high fat diet group.
Therefore, the in vivo anti-obesity studies of the HC revealed the following results.

The HC:
? reduces body weight gain,
? reduces food intake
? removes abdominal fat tissue
? causes thermogenesis
? improves the HDL levels
? restores glucose levels
? possesses liver protection property.

CLINICAL STUDY:
A prospective, randomized, double blind, placebo controlled clinical trial was carried out to evaluate efficacy and safety of HC in reducing the bodyweight in obese patients.
A total 30 numbers of subjects were included in the study as per the inclusion criteria. Subjects were randomized and equal number of subjects were assigned to HC treatment group A and placebo treatment group B. The clinical study was conducted for 56 days at Sampoorna Healthcare and Amruth Polyclinic, Bangalore, India. Subjects were on a treatment for 56 days and examined at 0th, 2nd, 4th and 8th week of the study. The subjects were asked to take one tablet three times a day for 08 weeks before breakfast and meals. The tablet for treatment group A contained 500 mg of Herbal composition of the present disclosure. The tablet for treatment group B contained 500 mg of placebo. Subjects followed up for two weeks after treatment period that was 10th week of the study.
? On 0th week, subjects were examined for physical examination, anthropometric measurements, biochemical parameters, HbA1c measurements and hematology parameters as baseline data.
? During 2nd and 4th visits, anthropometric measurements, physical examination and any adverse events were reported.
? On 8th week visit, physical examinations, anthropometric measurements, lipid profile, fasting blood glucose, HbA1c, quality of life assessment, adverse events, concomitant medications were recorded. All subjects were counseled for diet and exercise compliance at every visit.
? 10th week visit was meant to examine vital signs, physical examination, recording of adverse effects, serious adverse events, concomitant medications, weight, Body mass index & waist circumference, waist to hip ratio and waist to height ratio.

The results of the clinical study are provided in Table No. 19 and Table No. 20
Table 19: Results of clinical study of the group treated with Herbal Composition of the present disclosure
No. Parameters Visit 1 Visit 4 Difference in %
1 Body weight(kg) 82.5±12.3 77.25±12.9 6.36
2 Body mass index
(BMI kg/m2) 30.96±1.76 28.92±2.0 6.59
3 Waist circumference (cm) 101.25±6.67 94.92±5.9 6.25
4 Hip circumference
(cm) 106.33±2.05 101.91±1.67 4.16
5 Triglycerides
(mg/dL) 168.5±73.56 143.83±46.13 14.64
6 Cholesterol
(mg/dL) 214.08±33.2 187.91±21.2 12.22
7 LDL
(mg/dL) 138.27±34.9 104.33±25.73 24.55
8 VLDL
(mg/dL) 33.7±14.71 28.76±9.22 14.66
9 HDL
(mg/dL) 42.1±8.4 54.81±5.2 30.19

Table 20: Results of clinical study of the of Placebo treated group
No. Parameters Visit 1 Visit 4 Difference in %
1 Body weight(kg) 85.5±10.9 84.95±10.88 0.643
2 Body mass index
(BMI kg/m2) 32.33±1.88 32.04±1.81 0.90
3 Waist circumference
(cm) 104.79±5.36 104.42±5.33 0.35
4 Hip circumference
(cm) 108.83±1.70 108.46±1.73 0.34
5 Triglycerides
(mg/dL) 188.5±75.4 185.75±74.68 1.46
6 Cholesterol
(mg/dL) 204.91±43.8 201.16±43.2 1.83
7 LDL
(mg/dL) 108.85±59.7 107.75±59.16 1.01
8 VLDL
(mg/dL) 37.7±15.08 37.15±14.93 1.46
9 HDL
(mg/dL) 41.92±4 41.93±3.52 0.02
Clinical data suggests that Herbal Composition of the present disclosure is safe and is significantly efficient to reduce body weight and other obesity related complications.

ECONOMIC SIGNIFICANCE AND TECHNICAL ADVANCEMENT
The herbal composition of the present disclosure:
? Is safe and nontoxic;
? Reduces food intake;
? Reduces body weight and body weight gain;
? Removes abdominal fat tissue;
? Removes BAT;
? Causes thermogenesis;
? Improves HDLc levels;
? Restores glucose levels;
? Possesses liver protection efficiency; and
? Possesses good antioxidant properties.
Based on the above results herbal composition was found to be safe and has shown good antiobesity activity both by preclinical and clinical studies.

Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the invention to achieve one or more of the desired objects or results.

While certain embodiments of the inventions have been described, these embodiments have been presented by way of example only, and are not intended to limit the scope of the invention. Variations or modifications to the composition/formulation of this invention, within the scope of the invention, may occur to those skilled in the art upon reviewing the disclosure herein. Such variations or modifications are well within the spirit of this invention. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of the invention.

The numerical values given for various physical parameters, dimensions and quantities are only approximate values and it is envisaged that the values higher than the numerical value assigned to the physical parameters, dimensions and quantities fall within the scope of the invention and the claims unless there is a statement in the specification to the contrary. ,CLAIMS:1. A process for the preparation of Mangifera indica extract; said process comprising the following steps:
i. obtaining plant material from Mangifera indica;
ii. treating the plant material by a plurality of operations selected from the group consisting of sorting, cleaning, washing, rinsing, drying, grinding, extracting and pulverizing to obtain treated plant material; and
iii. obtaining an extract of Mangifera indica comprising predetermined quantities of specific parts of Mangifera indica by an iterative extraction method using at least one solvent followed by concentrating and drying the extract.
2. The process as claimed in claim 1, wherein the plant material comprises a plurality of parts of Mangifera indica selected from the group consisting of leaves, flowers, seeds, stem, branches, bark, roots, skin of the fruit and a combination thereof.
3. The process as claimed in claim 1, wherein, the plant material is selected from the bark, leaves and skin of ripe fruit of Mangifera indica.
4. The process as claimed in claim 1 wherein the extract contains bark of Mangifera indica 60% to 90%, extract of dried leaves of Mangifera indica 5% to 20% and dried skin of ripe fruit of Mangifera indica 5% to 20%.
5. The process as claimed in claim 1 wherein the extract contains bark of Mangifera indica 80%, extract of dried leaves of Mangifera indica 10% and dried skin of ripe fruit of Mangifera indica 10 %.
6. The process as claimed in claim 4, wherein the extract after drying has moisture in the range of 1% to 3 %.
7. The process as claimed in claim 1, wherein the extraction is carried out by at least one technique selected from the group consisting of hot stirring, reflux conventional extraction method, percolation, decoction, and supercritical fluid extraction performed in any order.
8. The process as claimed in claim 1, wherein the solvent contains 60% to 80% ethanol and 40% to 20% water.
9. The process as claimed in claim 1, wherein the extraction the treated plant material is carried out at a temperature ranging from 70°C to 80°C and for a time period ranging from 5 to 10 hours.
10. The process as claimed in claim 1, wherein the extract is dried at a temperature ranging from 80°C to 85°C to obtain a dried extract.
11. A Mangifera indica extract obtained by the process as claimed in any of the preceding claims, wherein the extract comprises polyphenolic compounds comprising at least one of gallic acid, 3, 4-dihydroxy benzoic acid, methyl gallate, proplyl gallate, pyrogallol, parahydroxy benzoic acid, vanillic acid, syringic acid, ferulic acid and ethyl gallate; and flavonoids comprising at least one of mangiferin, catechin, epicatechin and quercetin dehydrate.
12. A herbal composition for managing obesity comprising :
Mangifera indica extract comprising 60% to 90% powdered extract of dried bark, 5% to 20% powdered extract of dried leaves and 5% to 20% of powdered extract of dried skin of ripe fruit said having moisture between 1% to 3%.
13. The herbal composition as claimed in claim 12, wherein the Mangifera indica extract comprises flavonoids and polyphenols.
14. The herbal composition as claimed in claims 12, wherein the flavonoids comprise at least one of mangiferin, catechin, epicatechin and quercetin dehydrate.
15. The herbal composition as claimed in claim 12, wherein the amount of flavonoids ranges from 27.63% to 29.49% of the total mass of the herbal composition.
16. The herbal composition as claimed in claim 12, wherein the polyphenols comprise at least one of gallic acid, 3, 4-dihydroxy benzoic acid, methyl gallate, proplyl gallate, pyrogallol, parahydroxy benzoic acid, vanillic acid, syringic acid, ferulic acid and ethyl gallate.
17. The herbal composition as claimed in claim 12, wherein the amount of polyphenols ranges from 17.1% to 20.7% of the total mass of the herbal composition.