INTRODUCTION:

The global prevalence of obesity has nearly doubled since 1980, which is predominantly because of new dietary habits and sedentary lifestyle. Changes in the diet seem to be the major driver of the rise of the global epidemic of obesity during the past 3-4 decades. [1] Obesity is associated with dyslipidemias, diabetes, musculoskeletal disturbances, particularly osteoarthritis, and some types of cancer, such as endometrial, breast and colon cancer [2, 3]

Obesity is defined when the body mass index (BMI) ≥30 kg/m2 and an imbalance occurs between the intake and expenditure of the energy. This identified a high growing health issue in the developed and the developing nations.[4]

Obesity is a metabolic syndrome which is also associated with the development of medical issues like cardiovascular disease (CVD), stroke, respiratory disorders, type 2 diabetes (T2DM), sleep apnea, some cancers, hypertension etc. [5-7]

Many medications have been used to manage obesity over the years, but most of them are now withdrawn due to their serious adverse effects [8]. Anti- obesity drugs have been approved and marketed but due to serious reported side-effects most of them have been withdrawn. Fenfluramine and Dexfenfluramine were withdrawn in 1990s from the market due to heart valve damage.[9]

Similarly in the year 2000 European Medicines Agency (EMA) have recommended the withdrawal of many anti-obesity drugs like mazindol, phentermine and diethylpropion. These medications were responsible for causing unfavorable risks greater than benefit ratio. [10]

Rimonabant a CB1 receptor blocker was not approved by US-FDA as it has reported increased in psychiatric side effects like anxiety, depression, suicidal conditions etc. [11]

The pathogenesis of obesity is complex as it involves the role of multiple strategies to solve the issue therefore a simple and complete drug therapy or formulation is much desirable and acceptable to the obese patients for treatment.[12]

In a study it was analyzed that between the years 1980 to 2015 approximately 603.7 million adults and 107.7 million children were found obese in the year 2015. [13]

In this context, plant species have become indispensable in providing extracts and isolated chemical compounds that serve as raw material for the development of obesity treatments. [14-17] Herbal supplements and diet-based therapies for weight loss are among the most common complementary and alternative medicine [CAM] modalities. [18] A wide variety of these natural products and medicinal plants, including crude extracts and isolated compounds from plants are being used to induce weight loss and prevent diet-induced obesity. Nowadays, these have been vastly used in management of obesity [19,20] due to their natural richness in phytocomponents with different anti-obesity and anti-oxidant effects, which directly affects the body metabolism and fat oxidation. Medicinal plants have been investigated on huge scale and have been reported to be useful in the treatment of obesity, diabetes and other chronic diseases related to it. [21]

Trachyspermumammi, syn. Carumcopticum, belonging to family Apiaceae, commonly known as ajowan, is a glabrous annual plant. Its fruits are aromatic, with hollow stems, striated and much branched. [22] The fruit possesses stimulant, antispasmodic and carminative properties. It is an important remedial agent for flatulence, atonic dyspepsia and diarrhoea. [23] The seed of ajowan is bitter, pungent and it acts as anthelmintic, carminative, laxative, and stomachic agent. It also cures abdominal tumors, abdominal pains and piles. [24] The main component of this oil is thymol, which is used in the treatment of gastro-intestinal ailments and is the main therapeutic reason behind its anti-obesity property. Its lukewarm aqueous extract helps also to shed the weight gained post pregnancy. It is an important remedial agent for flatulence. [25-27]

Cinnamon Bark:

The most important constituents of cinnamon are cinnamaldehyde and trans-cinnamaldehyde, which are present in the essential oil, thus contributing to the fragrance and the various biological activities related with it. [28-31] Cinnamon extract from its bark in a type II diabetic animal model has been reported. The effect of cinnamon extract on anti-hyperlipidemia was evaluated with intake of 1, 3, or 6 g of cinnamon per day. It reduced serum glucose, triglyceride, LDL cholesterol, and total cholesterol. Hypolipidemic effect of cinnamaldehyde is mainly due to its influence on dietary fat absorption and cholesterol transportation. Aqueous extract of Cinnamon may act as a dual activator of PPARγ and α, and may be an alternative to PPARγ activator in managing obesity-related diabetes and hyperlipidemia.[32-34]

Pomegranate leaves:

Its leaves are rich in phytosterols, tocopherols and has unique fatty acid composition, mainly consisting of punicic acid (55 %), mainly in the pomegranate seed oil. Punicic acid, also known as trichosanic acid, is an omega-5 long chain polyunsaturated fatty acid and ia an isomer of conjugated α-linolenic acid with structural similarities to conjugated linoleic acid and α-linolenic acid [35-37] which are responsible for the anti-atherosclerotic and serum lipid-lowering activities.[38] Cinnamon has got antihyperlipidemic effect also. [39]

Garlic buds:

Some of the sulfur compounds present in garlic such as illicit, ajoene, S-allylycysteine, diallyldisulfide, Smethylcysteinesulfoxide, and Sallylcysteinesulfoxide, which are considered to be responsible for the therapeutic properties of garlic. [40] Its hypolipidemic actions 41 or lipid-lowering effects may occur via inhibition of HMG-CoA reductase or other enzymes, possibly due to diallyl di- and trisulphide components of garlic.[42-44] A large number of reports demonstrate positive impact of chronic use of garlic on lowering of plasma lipids.45 Epidemiologic studies have suggested that fresh garlic has lipid-lowering activity. Long-term dietary supplementation of fresh garlic may exert a lipid-lowering effect partly through reducing intestinal MTP gene expression, thus suppressing the assembly and secretion of chylomicrons from intestine to the blood circulation.[46] Another short-term supplementation of garlic in human subjects has demonstrated an increased resistance of LDL to oxidation. These data suggest that suppressed LDL oxidation may be one of the powerful mechanisms accounting for the hypolipidemic properties of garlic.[47] Studies have revealed that Garlic (1–4% in diet) and garlic protein administration in hypercholesterolemic rats induced by a high-cholesterol diet, significantly reduced serum cholesterol, triglyceride and LDL cholesterol.[48-54]

Black pepper fruits:

It belongs to family Piperaceae. The plant contains phenols, various derivatives of lignans, terpenes, flavonoid, alkaloid, chalcones, steroids, piperamine, piperolein B, sarmentosine, guineensine, pentadienoyls as piperidine, piperamide, piperettine, pipericide and piperine. [55-61] There are several research studies demonstrating remarkable effects of it against various diseases including cancer, inflammation, depression, diabetes, obesity and hepatotoxicity. [62]The fruits of the plant are widely used in traditional Mongolian medicine for treating hyperlipidemia.[63- 66]

MATERIALS AND METHODS:

All the constituents of the formulation as mentioned in Table 1 such as fruits of ajowan, bark of cinnamon, buds of garlic, fruits of black pepper were procured from the local market of Kanpur, whereas leaves of pomegranate were obtained from the herbal garden of PSIT, Kanpur. These crude drugs were cleaned thoroughly, shade dried and further processed to obtain the extracts.

Table 1 Constituents of the polyherbal formulation

S. No.	Part of the plant	Common name	Botanical name
1.	Fruits	Ajowan	Trachyspermumammi
2.	Bark	Cinnamon	Cinnamomum zeylanicum
3.	Leaves	Pomegranate	Punica granatum
4.	Buds	Garlic	Allium sativum
5.	fruits	Black pepper	Piper nigrum

Preparation of the polyherbal formulation:

Alcoholic extracts were prepared for all the above mentioned plants, using soxhlet apparatus. The extracts were derived using the fruits of ajowan, bark of cinnamon, leaves of pomegranate, buds of garlic and fruits of black pepper. Three formulations PHE1, PHE2, PHE3 (polyherbal ethanolic extract) were prepared using these plants and the concentrations of the formulations were PHE1- 100 mg/kg, PHE2- 150 mg/kg and PHE3-200 mg/kg.

MATERIALS AND METHODS:

Group 1:

The animals received a standard pellet diet along with purified water for duration of 24 h over a period of 40 days and this was considered as a NC – Normal Control Group

Group 2:

The animals received a standard pellet diet along with high fat diet over a period of 40 days. This was considered as a HFD or High fat Diet Control Group

Group 3:

The animals received a standard pellet diet along with high fat diet over a period of 40 days. After 1 week of study the animals were administered with a standard dose of Or list at 5 mg/kg b. wt. which was continued until the completion of experiment.

Group 4:

The animals received a standard pellet diet along with high fat diet over a period of 40 days. After 1 week of study the animals were administered with a test dose of PHE Extract 100 mg/kg b. wt. which was continued until the completion of experiment.

Group 5:

The animals received a standard pellet diet along with high fat diet over a period of 40 days. After 1 week of study the animals were administered with a test dose of PHE Extract 150 mg/kg b. wt. which was continued until the completion of experiment.

Group 6:

The animals received a standard pellet diet along with high fat diet over a period of 40 days. After 1 week of study the animals were administered with a test dose of PHE Extract 200 mg/kg b. wt. which was continued until the completion of experiment.

The animals (male wistar rats) weighing 150–170 g were divided into six groups where n=6 in each group. The rats were fed with standard pellet in normal control. Whereas other groups received HFD (High Fed Diet) during the entire course of experiment of 40 days. The rats received water and food ad libetum. For testing the activity of the poly herbal extract three concentrations of extract (100, 150 and 200 mg/kg b. wt.) were administered orally to three groups for 40 days. All the experiments involved the use of laboratory animals following the Institute Animal Ethics Committee regulations duly approved by committee. The college IAEC number is 1273/PO/Re/S/09/CPCSEA for Research for Education Purpose on small animals

Group I: Normal control group (NCG)

Group II: High fat diet group (HFD)

Group III: HFD + Orlistat 5 mg/kg b. wt.

Group IV: HFD +PHE Extract 100 mg/kg b. wt.

Group V: HFD + PHE Extract150 mg/kg b. wt.

Group VI: HFD +PHE Extract 200 mg/kg b. wt.

The experimental design used in the study is as mentioned in Table 1

Table 1: Experimental design:

S. No.	Group Number	Group Name	Dosing	Number of Animals
1.	Group I	NCG	Normal control group (NCG)	6
2.	Group II	Control Group	High fat diet group (HFD)	6
3.	Group III	Standard Group	HFD + Orlistat 5 mg/kg b. wt.	6
4.	Group IV	PHE-1	HFD + PHE Extract 100 mg/kg b. wt.	6
5.	Group V	PHE-2	HFD + PHE Extract 150 mg/kg b. wt.	6
6.	Group VI	PHE-3	HFD + PHE Extract 200 mg/kg b. wt.	6

OBSERVATION:

Statistical Analysis:

The results were expressed in mean ± SD (Standard Deviation) and the statistical analysis was conducted by one-way ANOVA using SPSS, Version 18. The values with p < 0.05 were considered as statistically significant.

Determination of Body Weight:

In the entire course of 40 days experiment the weight of rats were recorded from day one followed by monitoring of body weight weekly. For assessment of the effect of treatment on body weight the weight of all animals were recorded on last day of experiment and the mean was calculated for all groups.

The weight of all groups were taken at the end of 40 days and mean weight from all groups are presented in the Table 2 and Figure 1 as well as in a chart to get an estimation of the body weight of all groups.

Table 2: Effect of HFD and Treatment on Body Weight of Wistar Rats

S. No.	Group Number	Dosing	Body Weight
1.	Group I	NC	424 ± 16^a
2.	Group II	HFD	495 ± 25^bc
3.	Group III	HFD + Orlistat	442± 21^b
4.	Group IV	HFD + PHE Extract (1) 100 mg/kg b. wt.	448 ± 15^ac
5.	Group V	HFD + PHE Extract (2) 150 mg/kg b. wt.	440 ± 24^b
6.	Group VI	HFD + PHE Extract (3) 200 mg/kg b. wt.	435 ± 26^bc

The rats were fed with normal diet in NC (Normal Control Group) and HFD (High fat Diet) in Groups II, II, IV, V and VI for a duration of 40 days. (n=6 for each group) The values from all 6 rats are expressed as mean ± standard deviation (p < 0.05) ^aSignificantly different from the control, ^bSignificantly different from control group with HFD, PHE 1, 2, 3 (Poly Herbal Ethanolic extract). The data is analyzed by parametric method-ANOVA.

Figure 1: Effect of HFD and Treatment on Body Weight of Wistar Rats

Figure 2: A graphical representation of the Effect of Treatment on the fecal weights and fecal lipid content in all groups

Determination of fecal weight and lipid profile of the fecal matter:

The fecal weight through day1 of the experiment till of last day of experiments were weekly monitored and tabulated as mean values to draw a comparison of the effect of treatment during the entire course. The lipid content from the fecal matter was also calculated to find out the effect of the treatment in all groups therefore determining the lipid values.

The table 3 and figure 2 shows the fecal matter and fecal lipid content on initial day and at the end of 40 days research study.

Determination of Fasting Blood Glucose:

The entire experiment was performed for 40 days and the fasting blood glucose level was recorded on day 1 and day 30 of the experiment. The mean fasting blood glucose was calculated and the observations were plotted and compared in the table 4 and figure 3.

Table 3: The Effect of Treatment on the fecal weights and fecal lipids

S. No.	Group Number	Dosing	Fecal Matter (Initial)(mg/g)	Fecal Matter (Final) (mg/g)	Fecal Lipid (Initial)(mg/g)	Fecal Lipid (Final)(mg/g)
1.	Group I	NC	1.1 ± 0.11	1.3 ± 0.16	8 ± 3.35	9.9 ± 0.29
2.	Group II	HFD	1.2 ± 0.101	1.9 ± 0.061^a*	12 ± 2.43	13.1 ± 2.2^a*
3.	Group III	HFD + Orlistat	1.3 ± 0.13	1.0 ± 0.58^b*	12.5 ± 1.2	17.1 ± 0.5^b*
4.	Group IV	HFD + PHE Extract 100 mg/kg b. wt.	1.3 ± 0.17	1.4 ± 0.42^NS	11.3 ± 1.5	15.6 ± 3.5^NS
5.	Group V	HFD + PHE Extract 150 mg/kg b. wt.	1.2 ± 0.025	1.2 ± 0.12^b*	12.1 ± 0.31	16.2 ± 0.18^b*
6.	Group VI	HFD + PHE Extract 200 mg/kg b. wt.	1.3 ± 1.11	0.99 ± 0.6^b*	12.5 ± 2.02	17.9 ± 2.08^b*

The data is analyzed by parametric method-ANOVA.

The data’s presented are given as mean ± S.D (n = 6).The values are statistically significant with *p < 0.05^.

^aSignificantly different from NC,

^bSignificantly different from HFD control, HFD treated and standard groups

Table 4: Blood glucose levels before and after treatment in Control group, Standard Group and Intervention groups (different concentration extracts ofPoly Herbal Preparation) in wistar rats

S. No.	Group Number	Dosing	Blood Sugar (mg/dl) (Day 1)	Blood Sugar (mg/dl) (Day 30)
1.	Group I	NC	70.7±2.2	70.2 ± 3.4
2.	Group II	HFD	74.2±1.1	158.1 ± 7.6^a*
3.	Group III	HFD + Orlistat	72.3±1.6	95.1 ± 1.2^b*
4.	Group IV	HFD + Extract 100 mg/kg b. wt.	73.9±1.9	100.9 ± 2.1^NS
5.	Group V	HFD + Extract 150 mg/kg b. wt.	71.2±2.4	98.7 ± 2.1^b*
6.	Group VI	HFD + Extract 200 mg/kg b. wt.	72.1±1.3	91.2 ± 3.4^b*

The data is analyzed by parametric method-ANOVA.

The data’s presented are given as mean ± S.D (n = 6). The values are statistically significant with *p < 0.05^.

^a Significantly different from NC,

^bSignificantly different from HFD control, HFD treated and standard groups

NS: Non-Significant

Figure 3: A graphical representation of the Mean fasting Blood glucose levels before and after treatment in Control group, Standard Group and Intervention groups (different concentration extracts of Poly Herbal Preparation) in wistar rats

Table 5: A comparison of lipid profiles in in Control group, Standard Group and Intervention groups (different concentration extracts of Poly Herbal Preparation) in wistar rats

S. No.	Group Number	Dosing	TC	TG	LDL	HDL	VLDL (mg/dl)
1.	Group I	NCG	60 ± 5^a	61 ± 17^a	40 ± 6^a	14.1 ± 1.3^a	16.38 ± 1.15
2.	Group II	HFD	74 ± 10^b	76 ± 23^b	58 ± 23^b	18.5 ± 2.4^a	28.29 ± 4.34
3.	Group III	HFD + Orlistat	62± 5^a	63 ± 27^ab	44 ± 6^ab	14.6 ± 1.5^a	19.32 ± 1.67**
4.	Group IV	HFD + Extract 100 mg/kg b. wt.	65 ± 7^a	66 ± 14^a	47 ± 8^a	14.9 ± 2.1^a	23.14 ± 1.24*
5.	Group V	HFD + Extract 150 mg/kg b. wt.	63 ± 6^ab	64 ± 15^ab	45 ± 9^ab	14.5 ± 1.8^a	20.5±1.13
6.	Group VI	HFD + Extract 200 mg/kg b. wt.	60 ± 7^a	62 ± 12^a	41 ± 5^a	14.3 ± 2.5^a	17.46 ± 1.55**

The data is analyzed by parametric method-ANOVA.

The data’s presented are given as mean ± S.D (n = 6). The values are statistically significant with *p < 0.05^.

^a Significantly different from NC,

^bSignificantly different from HFD control, HFD treated and standard groups

Determination of Serum Lipid Profiles:

The serum lipid values like TC, TG, LDL, HDL and VLDL were calculated and tabulated for all treatment, control and standard groups. The blood samples were collected from all rats in a group and respectively it was done for all groups.

The blood samples collected were centrifuged at 2500 rpm/min for a duration of 15 minutes and serum was separated which was stored at -80⁰ C for all the biochemical analysis.

Now the serum lipid profile values were calculated by using auto-analyzer from Sigma Aldrich to draw a comparison between the lipid values from all groups. The data’s are as tabulated and plotted in table 5 and figure 4 respectively

Figure 4: A comparison graph of lipid profiles in in Control group, Standard Group and Intervention groups (different concentration extracts of Poly Herbal Preparation) in wistar rats

Comparison of three groups given different concentrations of the herbal mixture extracts on obesity-related parameters in HFD-fed rats.

DISCUSSIONS:

Effect of different concentrations of Poly Herbal Ethanol extracts on body weight:

On feeding the rats with HFD resulted in an increase in the body weight of all wistar rats. However those groups which were administered with Standard drug Orlistat or with variable concentrations of poly herbal ethanol extract prepared were showing less increase in body weight compared to the HFD only fed animal group. The poly herbal extract with concentration of 200 mg/kg b. wt. showed the maximum control in body weight which is a good control in case of obesity. A poly herbal formulation which can control the weight of an obese animal can prove to be a potential source for treating obesity disorders.

Effect of different concentrations of Poly Herbal Ethanol extracts on fecal weight and fecal lipids:

The fecal weight and fecal lipid profile was monitored for all groups and mean values were analyzed. The lipid content in fecal output was found to be gradually increasing in groups with increased dosage. A significant (p < 0.05) increase in lipid content in fecal matter was reported with Group VI being the highest which was receiving Poly herbal ethanol extract with highest concentration 200 mg/kg b. wt. The fecal output of the treated groups was gradually lowered in comparison to the HFD group.

Effect of different concentrations of Poly Herbal Ethanol extracts on Fasting Blood Glucose levels:

The oral administration of the poly herbal extracts in different concentrations to respective groups has proved to lower the fasting blood glucose levels. The extract has reversed the glucose levels to normal values even after the administration of HFD. In the tabulated data the herbal extract with highest concentration administered was found to be significantly effective in reducing the blood glucose levels.

Effects of different concentrations of Poly Herbal Ethanol extracts on serum lipid profiles:

The rats were checked for the serum lipid profiles after monitoring the scheduled diet and treatments respectively. The serum lipid profile values were found to be significantly low in case of treated groups as compared to HFD group. The poly herbal extract treated 100mg/kg extract dose showed a lesser effect on the lipid profile whereas the 200mg/kg dose extract from the poly herbal preparation proved to be most effective in lowering down the values of TG, TC, VLDL, HDL and LDL. The administration of Poly Herbal Ethanol Extracts with 100 mg/kg b wt., 150 mg/kg b wt. and 200 mg/kg b. wt. has proved significantly (p < 0.05) for normalizing the levels of serum lipid profiles.

CONCLUSION:

Obesity is considered as an incurable disorder which directly increases the risk cardiovascular diseases and diabetes. Lifestyle affects the weight of a person which directly leads to obesity and other associated complications. A large section of the world’s population is suffering from long term obesity issues. Despite of promising results in the weight reduction and improvement of metabolism lately many effective drugs have been withdrawn from the market due to their reported adverse effects.

Therefore need of an herbal home remedy or medicine from indigenous sources could prove as an effective cure for treating obesity issues which have not proven to cause any side effects so far. The research experiment we performed included the use ethanol extract from five herbal sources and used in the treatment of obesity induced wistar rats.

Comparison of effectiveness of the extracts of variable concentrations were performed in the experiment where the extract with highest concentration provide out to be most effective in treating obesity in HFD included wistar rat groups.

The alcoholic extract of herbal powder of Ajowan fruit, Cinnamon bark, Leaves of pomegranate, Garlic buds and dried fruits of black pepper was found to be most effective at the concentration of 200 mg/kg b. wt. in reducing serum lipid values, maintain the blood sugar, reducing the fecal output and increasing the lipid content in fecal output altogether contributing in controlling all parameters which are found be increased in cases of obesity as also responsible for causing obesity associated complications like cardiovascular disorders and diabetic issues.

CONFLICT OF INTERERST:

The authors have declared no conflicts of interest.

ACKNOWLEDGEMENTS:

We are very thankful for Dr. A. K Rai, Director Pharmacy, Pranveer Singh Institute of Technology for guiding us throughout our journey of research.

FUNDING DECLERATION:

There were no funding declarations.

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Received on 11.12.2018 Modified on 21.01.2019

Research J. Pharm. and Tech. 2019; 12(4): 1857-1864.

DOI: 10.5958/0974-360X.2019.00310.X