Haque MR, Ansari HS. Anti-Obesity Effect of Arq … Drug Res 2018; 68: 637–647

Original Article Thieme

IntroductionObesity has become a social health problem in the world. It is a complex and heterogeneous disorder presently affecting more than 100 million people worldwide and causing serious socio-eco-nomic problems [1]. Obesity is a chronic and expensive disease that is rarely curable and is increasing in prevalence in most of the world. The prevalence of obesity is rising both in developed and develop-ing countries [2]. The prevalence of obesity in the United States has increased; presently, one-third of American adults are considered obese. It is estimated to cause between 280,000 and 325,000 deaths per year in the United States. Delays in treatment increase in risk of future development of diabetes and its related, and of heart disease. Among other factors, obesity is an important con-

tributor to develop various chronic diseases in humans. Data from the Framingham Heart Study suggest that 78 % of essential hyper-tension in men and 65 % of essential hypertension in women can be directly attributed to obesity [3]. Obesity is accompanied by ex-cess lipid accumulation, impaired glucose tolerance, and elevated serum triacylglycerol (TG) concentration; thus, it is positively asso-ciated with the progression of various chronic diseases such as type 2 diabetes mellitus, dyslipidemia, insulin resistance, cardiovascular diseases (hypertension, stroke and atherosclerosis), osteoarthritis, sleep apnea, gallstones, fat liver disease and cancers [4]. In past years, many numbers of drugs have been approved for the treat-ment of obesity; however, most of them have been withdrawn from the market because of their adverse effects. In fact, amphetamine,

Haque MRafiul, Ansari HS. Anti-Obesity Effect of Arq … Drug Res 2018; 00: 00–00

Anti-Obesity Effect of Arq Zeera and Its Main Components Thymol and Cuminaldehyde in High Fat Diet Induced Obese Rats

AuthorsMohammad Rafiul Haque1, 2, H. Shahid Ansari1

Affiliations1 Department of Pharmacognosy and Phytochemistry,

Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi, India

2 HIMT College of Pharmacy, Greater Noida, Uttar Pradesh, India

Key wordsArq zeera, thymol, cuminaldehyde, obesity, high fat diet, orlistat

received 20.12.2017 accepted 09.03.2018

BibliographyDOI https://doi.org/10.1055/a-0590-1956Published online: 10.4.2018 Drug Res 2018; 68: 637–647© Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379

CorrespondenceMd Rafiul HaqueHIMT College of Pharmacy, Knowledge park1Gr. Noida(U.P)Pin code - 201310Tel.: + 91/886/0520 086 mdrhaque79@gmail.com

AbStR Act

Arq zeera is a distillate product that prepared from four differ-ent herbs namely Trachyspermum ammi L., apiaceae, Zingiber officinale Roxb., zingiberaceae, Carum carvi L.,apiaceae and Cuminum cyminum L., apiaceae. The present study aims to determine the antiobesity effect of arq zeera and its main com-ponents thymol and cuminaldehyde in high fat diet induced obese rats and to explore its mechanism of action. In current study, orlistat was used as positive controls. Male Wistar rats were fed with HFD for 42 days to induce obesity. HFD-fed rats were administered with arq zeera, thymol, cumic aldehyde, thymol + cuminaldehyde and orlistat for 28 days. During the course of treatment, body weight and food intake frequently observed and after end of treatments, liver weight, visceral fat pad weight, plasma lipid proflie, alanine aminotransferase, as-partate aminotransferase, glucose, insulin, leptin levels and pancreatic lipase activity were studied on all treated obese rats. The histopathology of liver was also studied. After the treat-ments of arq zeera and its main components, body weight, food intake, liver weight, visceral fat pad weight and the level of lipid profile, alanine aminotransferase, aspartate aminotran-ferase, glucose, insulin, and leptin were found to be decreased and pancreatic lipase inhibition were increased. Arq zeera showed more potential antiobesity effect than orlistat. Accord-ing to our present findings, arq zeera and its main components possessed potent antiobesity effect on high fat diet -induced obese rats and excreted anti-obesity effect partly via hypolipi-demic, hypoglycemic, hypoinsulinemic, hypoleptinemic and pancreatic lipase inhibition action.

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Haque MRafiul, Ansari HS. Anti-Obesity Effect of Arq … Drug Res 2018; 68: 637–647

Original Article Thieme

fenfluramine, dexfenfluramine, rimonabant and sibutramine licens-es have been withdrawn due to an increased risk of psychiatric dis-orders, valvular heart disease, pulmonary hypertension and non-fatal myocardial infarction, or stroke. Even if orlistat is not as effec-tive as other drugs in reducing body weight, orlistat is presently the only available choice for the treatment of obesity because of its safety for cardiovascular events and positive effects on diabetic control [5]. That is why, in the current study, orlistat was used as standard drug. Pharmacological treatment of obesity has been a particularly challenging task. Most of the drugs have failed and fell into disrepute either due to ineffectiveness or adverse effects [6]. Because of the adverse effects associated with these anti-obesity drugs, many trials have been recently conducted to find and devel-op new antiobesity drugs through herbal medicines or chemical compounds from herbals that would minimize the side effects. Nu-merous animal studies and clinical studies with various herbal med-icines have been performed, and some studies reported significant improvements in controlling body weight without any noticeable adverse effects [7]. Ayurvedic and Unani medicines have been used for treatment of obesity for centuries. However, many of them have not been tested for efficacy and safety using modern scientific methods. Arq zeera is one of the most well-known traditional herb-al medicines frequently used to treat obesity in Unani System of In-dian Medicine. The preparations marketed by different manufac-turers have minor differences in their composition ratio. These preparations are thought to alter fat metabolism, and increase basal metabolic rate. However, scientific data is not available for these claims. Polyherbal (mixture of herbs), as the basic composi-tion unit Unani compound formulas, have a special clinical signifi-cance in Unani system of Indian Medicine. Polyherbal formulations more willingly than monotherapeutic herbal formulation are fre-quently used because of the synergistic effect. Many polyherbal formulations such as Safoof -e- Muhazzil, OB-200 G Lipovedic etc. have revealed their efficacy and potency against obesity [8–10]. Arq zeera, a polyherbal distillate product of four herbs such as Ajwain (Trachyspermum ammi Linn. Apiaceae, fruits), Ginger rhi-zome (Zingiber officinale Roxb. Zingiberaceae, rhizome), black car-away (Carum carvi L. Apiaceae, fruits) and cumin (Cuminum cyminum L. Apiaceae, fruits) and these herbs are also the main con-tained in many traditional Unani and Ayurvedh compound formu-las such as Qurs-e-Pudina (composed of eight herbs, i. e., Trachy-spermum ammi, Zingiber officinale, Carum carvi, Cuminum cyminum, Mentha arvensis, Foeniculum vulgare, Cinnamomum Zeyleniculum, Piper nigrum and Safoof -e- Muhazzil (composed of seven herbs, i. e., Tachyspermum ammi, Carum carvi, Rosa dema-scena, Nardostachys jatamansi, Origanum vulgare, Foeniculum vul-gare, and Ruta greveolense or some modification in formulae has the effect of stomachic, carminative, digestive and antiobesity ef-fect respectively [11, 12]. In the Indigenous systems of medicine practiced in Southeast Asia, such as Siddha, Ayrveda and Unani sys-tems, these four herbs such ajwain, ginger cumin and caraway seeds are prominently considered carminative, eupeptic, antispas-modic, astringent and used in the treatment of mild digestive dis-orders, diarrhea, dyspepsia, flatulence, morning sickness, colic, dys-peptic headache,bloating and obesity and are said to promote the assimilation of other herbs and to improve liver function [13, 14]. It has been reported that thymol, a biomarker monoterpenic phe-

nolic compound of Tachyspermum amm has anti-oxidant, anti-mi-crobial, anti-inflammatory activity, blood pressure lowering action and hypocholestrolemic action through inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity [15–18]. Cumi-naldehyde, a quality biomarker compound of Cuminum cyminum L and Carum carvi L., has been reported to have, anti-inflammato-ry activity, anti- oxidant activity, anti-hyperglycemic effect and an-tihyperlepidemic effect [19–21].

In the Unani system of Indian medicine, arq zeera has been tra-ditionally used alone or combination with other herbal product for the treatments of obesity. But, the underlying anti- obesity mech-anism of arq zeera has not been elucidated so far. Therefore in cur-rent article, effects of arq zeera and its main components thymol and cuminaldehyde on obese rats induced by high fat diets (HFD) were studied and then their synergetic effect was also evaluated. The preliminary anti-obesity mechanism of arq zeera was also dis-cussed on the basis of their investigation. The results obtained from arq zeera were also compared against standard drug orlistat.

Materials and Methods

ChemicalsandkitsPhytochemical reference compounds thymol (purity; 95.5 %, Mo-lecular Weight; 150.22), cuminaldehyde (purity; 98 %, Molecular Weight; 148.20) 2, 2-diphenyl-picrylhydrazyl (DPPH), suphuric acid, anisaldehyde, ethanol, vanillin, and acetic anhydride were pur-chased from Sigma–Aldrich (St. Louis, MO, USA). All contamination standards like toxic metals, pesticide, aflatoxins were procured from Spectra Laboratory, Okhla, phase II, New Delhi) AST, ALT, glucose, total cholesterol (TC), and total triglycerides were purchased from Span Diagnostics Ltd, Surratt, Gujarat, India) and high-density lipo-protein-cholesterol (HDL-C) from Reckon Diagnostics Pvt Ltd, Baro-da, Gujarat, India). Insulin ELISA kit (Crystal Chem. Inc., Downers Grove, IL 60515, USA), rat leptin ELISA kit (BioVendor, Czech Repub-lic), Quanti ChromTM Lipase Assay Kit (DLPS-100), (Corporate Place, Hayward, CA 94545, USA) and orlistat (purity > 99.5 %) were obtained from Labex corporation, vasant kunj, New Delhi).

HerbalmaterialsAll the ingredients were collected from Unani raw drug dealers with the knowledge of Unani physician at favorable environmental con-dition at its suitable seasons and identified by expert botanical tax-onomist Dr. H. B. Singh (scientist B), from National Institute of Sci-ence Communication and Information Resources (NISCAIR), New Delhi, India. Voucher specimen and identification certificate refer-ence number NISCAIR/RHMD/Consult/2011-12/1753/53 was ob-tained and kept in the herbarium of National Institute of Science Communication and Information Resources (NISCAIR), New Delhi, India with voucher number No. 182045 for Trachyspermum ammi (L.) Sprague, fruits, No. 223456 for Zingiber officinale (Roxb.), rhi-zome, NO. 234564 for Carum carvi (L.), fruits and No. 304587 for Cuminum cyminum (L.), fruits future reference. Samples are also deposited in the herbarium of Pharmacognosy Department of Jamia Hamdard University with voucher number No. 0234 for for Trachyspermum ammi (L.) Sprague, fruits, No. 0223 for Zingiber officinale Roxb., rhizome, No. 0264 for Carum carvi (L.) fruits and No.

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Haque MR, Ansari HS. Anti-Obesity Effect of Arq … Drug Res 2018; 68: 637–647

0587 for Cuminum cyminum (L.), fruits. All the ingredients were au-thenticated by standardization parameters and compared with Phar-macopeial limits. All the ingredients were found under Pharmaco-peial limits and toxic metal (arsenic, lead, cadmium and mercury), aflatoxins (B1, B2, G1, and G2) and pesticide such as o, p- DDD, p, p- DDD, o, p- DDE, p, p- DDE, o, p- DDT, p, p- DDT, Endosulfan, α- HCH, β- HCH, γ- HCH, δ- HCH were not detected in the powder drugs.

MethodsofpreparationofarqzeeraCrushed the cleaned and dried all the four ingredients viz: Zingiber officinale (Roxb.), rhizome (125 g), Carum carvi (L.), fruits (125 g) Cuminum cyminum (L.) fruits (375 g) and Trachyspermum ammi (L.) Sprague, fruits (250 g) in an iron morter to obtained coarse powder and soaked in 12 L purified water. Transferred the soaked all four ingredient to the distillation plant along with purified water. Distilled the same at 100 ° C for about five and half hrs and collect-ed the 7.5 L of arq zeera [22].

Chemoprofileofformulationarqzeera byGCMSandGCThe Chemo profile of formulation was determined by GCMS and GC. The formulation arq zeera was dissolved in diethyl ether sol-vent and result in the formation of two layer, ether and aqueous layer. The ether extract was concentrated by using rotary evapora-tor (Buchi Rotavapor R-200) under vacuum and 0.2 μl of ether ex-tract subjected into GC and GCMS analysis for identification of chemical constituents [23]. The analysis of the volatile constitu-ents were run on a Shimadzu QP-2010 GC-MS system equipped with AB-Innowax 7031428 WCOT column (60 m x 0.25 mm x 0.25 μm) directly coupled to the MS. The carrier gas was helium with a flow rate of 1.21 ml/min. oven temperature was programmed as 50 °C for 1 min and subsequently held isothermal for 2 min. in-jector port: 250 °C, detector: 280 °C, split ratio 1: 50, volume in-jected: 1 μl of the oil. The recording was performed at 70 eV, scan time 1.5 s, mass range 40–750 amu. Software adopted to handle mass spectra and chromatograph was a Chem station. The gas chromatographic analysis of the volatile oil was carried out on Shi-madzu 2010 Gas Chromatograph (Japan) equipped with a flame ionization detector (FID) and AB-Innowax 7031428 WCOT fused capillary column (60 m x 0.25 mm x 0.25 μm). The injector and de-tector (FID) temperatures were maintain at 250 and 270 °C, respec-tively. The carrier gas used was nitrogen at a flow rate of 1.21 ml/min with column pressure of 155.1 kPa. The sample (0.2 μl) was in-jected into the column with a split ratio of 80:1. Component sepa-ration was achieved following a linear temperature programmed from 60–230 °C at a rate of 3 °C/min and then held at 230 °C for 9 min, with a total run time of 55.14 min. Percentage of the con-stituents were calculated by electronic integration of FID peak areas. The chemical constituents were identified by using NIST and Wiley library software and KI values. The GCMS and GC chromato-gram of arq zeera were shown in ▶Fig. 1–2.

AnimalsThe study was approved by the Institutional Animal Ethics Com-mittee (IAEC) of Hamdard University, New Delhi, which is registered with Committee for the Purpose of Control & Supervision of Exper-iments on Animals (CPCSEA), Government of India. Wistar male al-

bino rats, weighing 150 - 200 g, were procured from the Central Animal House Facility, Hamdard University, New Delhi for this val-uable study and acclimatized under standard laboratory conditions at 25 ± 2 °C, and relative humidity (50 % ± 15 %) and normal. The animals were kept in polypropylene cages under standard labora-tory conditions (12 h light and 12 h dark: day: night cycle) and had a free access to tap water ad libitum.

AnimalexperimentsAfter 7 days of acclimation, animals were randomly divided into seven groups (n = 6): one normal control group, one HFD control and remaining group as treatment groups. Animals in normal con-trol group were fed with normal pellet diet (12.5 % lipids, 62.3 % carbohydrate and 24.3 % protein; Amrut rat feed, Mfd by: Pranav Agro Industries Ltd, Maharashtra, India) while the other groups were fed with HFD (42 % lipids, 36 % carbohydrate and 22 % protein) ad libitum, respectively, throughout the experiment [24]. HFD was purchased from National Centre for Laboratory Animal Sciences (NCLAS), National Institute of Nutrition (NIN), Hyderabad, Andhra Pradesh, India). The human dose of arq zeera is being used 75 ml twice per day prescribed by Unani Physician. The dose of arq zeera for rats was calculated from human dose by using formula: Animal dose (ml/kg) = total human dose (75 ml twice a day) / average body wt (60 kg) multiplied by conversion factor (6.2) [25]. This formula-tion is considered safe at the dose of 75 ml when administered twice a day to the patient by an Unani physician [22]. Thymol and Cuminaldehyde dose were determined from the contents present

Thymol

Cuminaldehyde

▶Fig. 1 GC-MS chromatogram of arq zeera

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Haque MRafiul, Ansari HS. Anti-Obesity Effect of Arq … Drug Res 2018; 68: 637–647

Original Article Thieme

in the arq zeera. Normal control group received 0.5 % carboxy me-thyl cellulose (CMC) sodium aqueous solution. Thymol, orlistat and cumic aldehyde drugs were suspended in 0.5 % carboxymethyl cel-lulose sodium aqueous solution for animal’s treatment. AZ was also dissolved in 0.5 % CMC sodium aqueous solution to form suitable dose. Male Wistar rats were fed with HFD for 42 days to induce obe-sity. Treatments were started from 15th day and continued for four weeks. The treatment groups HFDF, HFDT, HFDC, HFDTC and HFDO were given arq zeera formulation, thymol, cuminaldehyde, thy-mol + cuminaldehyde and orlistat respectively at 7.75 ml/kg, 12 mg/kg, 6 mg/kg and 12 + 6 mg/kg twice per day and, 30 mg/kg of the body weight once per day of body weight by oral root [26]. During the course of treatment the treatment groups were contin-ued to feed with HFD. Body weight was measured weekly, body weight change measured from difference between final body weight and initial body weight. Food intake was estimated by sub-tracting the amount of food left in the cages from the total amount of food provided to each rat. Measured on day 1 & then on alter-nate day for 42 days in each group [27]. Blood was collected from the retro-orbital plexus of the all groups of overnight fasted rats using microcapillary tubes containing heparin on 43th day. Plasma was separated from blood by centrifugation (4000 rpm, 10 min) and transferred to Eppendorf tubes. The concentrations of glucose, total cholesterol (TC), triglycerides and high-density lipoprotein-cholesterol (HDL-C), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in plasma were measured with commercial

kits. Estimations of serum LDL cholesterol were measured by the using of Friedewalds equation [28]. And the assays of plasma insu-lin, leptin levels and pancreatic lipase activity were accomplished using the ELISA kit, according to the manufacturer’s protocol. All other chemicals used were of analytical grade. Double distilled water was used for all biochemical assays. After serum collection, all animals were killed by cervical dislocation; liver and visceral fat pad weights (mesenteric, perirenal and epididymal fats) were dis-sected out and weighed. And one rats liver from each groups i. e., seven rat liver were collected for histopathology study. The collect-ed rat liver tissue samples were fixed in 10 % formalin buffered so-lution, cut into 5 μm sections and stained with haematoxylin/eosin. The sections of liver tissues were studied to determine the level of tissue damage by HFD.

StatisticalanalysisStatistical analysis was carried out using Graphpad Prism 3.0 (Graphpad software, San Diego, California, USA). All of the data were expressed as mean ± SEM. Statistical analysis was performed by Dunnett’s t- test and p < 0.05 were considered to be significant.

Results

GCMSandGCanalysisThe GCMS and GC analysis of arq zeera were detected and identi-fied 37 chemical components. Thymol (44.40 %), Cuminaldehyde (21.13 %), 1-phenyl propanol (14. 234 %), and 4-ethyl-3-nonen-5-yne (11.29 %) were major chemical components which comprised about 91 % among 37 compounds shown in ▶table 1.

Theeffectofformulationarqzeeraanditsmaincomponentsonbodyweight,foodintake,visceralfatpatandliverweightHFD group (pathogenic group), showed significant (p < 0.01) in-crease in body weight gain than those of the normal healthy con-trol group (basal), after 6 week of treatment. HFDF, HFDT, HFDC, HFDTC and HFDO groups were significantly (p < 0.01) reduced the body weight as compared to HFD group shown in ▶table 2. Food intake, visceral fat pat and liver weight in HFD group were higher than that of normal healthy control group. HFDF, HFDT, HFDC, HFDTC and HFDO were significantly (p < 0.001) reduced the viscer-al fat pat and liver weight as compared to HFD group. HFDF groups was significantly (P < 0.01) reduced the food intake and HFDT and HFDTC showed less significant (p < 0.05) while HFDC and HFDO groups did not show any considerable reduction the food intake shown in ▶table 3.

TheeffectofformulationarqzeeraanditsmaincomponentsonSerumlipidprofilePlasma TC, TGs, and LDL cholesterol, in HFD group were significant-ly elevated as compared to basal (normal healthy group) while HDL-cholesterol level was significantly reduced in HFD group as com-pared to control group. HFDF, HFDTC and HFDO were significantly (p < 0.001) reduced the raised level of TC, TGs, and LDL-cholesterol and increase the level of HDL-cholesterol as compared to HFD

Thymol

Cuminaldehyde

▶Fig. 2 GC- chromatogram of arq zeera

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Haque MR, Ansari HS. Anti-Obesity Effect of Arq … Drug Res 2018; 68: 637–647

▶table 1 Chemo profile of formulation Arq Zeera.

S.no KI value compound Area %

1. 986 2-Methyl-propane-1-one 0.0435

2. 1012 N-Pentadecanoic Acid 0.1712

3. 1025 D-Limonene 0.0598

4. 1028 1,8-Cineol 0.01886

5. 1029 O-Cymene 2.1478

6. 1033 2-Heptanol 0.0407

7. 1035 alph -Terpinene 2.110

8. 1048 p-Cresol 0.0561

9. 1135 Limonene epoxide 0.1003

10. 1171 4-Terpineol 0.1990

11. 1175 α-Terpineol 0.0503

12. 1177 4-Thujanol 0.0211

13. 1202 Borneol 0.1318

14. 1182 4-Isopropyl-1-methyl-2-cyclohexen-1-ol 0.2260

15. 1189 4-p-Menthadien-7-ol 0.2596

16. 1224 Sabinyl-acetate 0.0412

17. 1226 β.-Citronellal 0.0969

18. 1239 p-Cumic aldehyde 21.1329

19. 1238 Hexan-2-ol 0.2663

20. 1242 Trans-Citral 0.0521

21. 1251 Cuminol 0.4101

22. 1255 Nerol 0.0411

23. 1256 1-Phenylpropanol 14.234

24. 1258 Geraniol 0.054

25. 1280 4-Ethyl-3-Nonen-5-Yne 11.296

26. 1287 4-(Isopropyl phenyl) Methanol 0.0411

27. 1294 Thymol 44.404

28. 1306 Carvacrol 0.2149

29. 1334 2-Heptanol 0.0407

30. 1393 4-Isopropyl-2-cyclohexen-1-yl)methanol 0.0412

31. 1438 β-Linalool 0.1150

32. 1477 α-Curcumin 0.0784

33. 1492 Zingiberene 0.2595

34. 1495 β-Bisabolene 0.3058

35. 1508 β-Sesquiphellandrene 0.0820

36. 1514 Farnesene 0.0970

37. 1562 Nerolidol 0.0239

Identification Methods = MS, RI (KI),

▶table 2 The results of the effect of formulation and its components on the body weight.

Groups Initial wt (g) 14th day 43rd day Weight gain during treatment (g)

Control 155.2 ± 2.166 180.87 ± 4.34 251.08 ± 3.12 70.21 ± 5.12

HFD 158. 32 ± 1.261 212.56 ± 3.56 342.45 ± 10.2 129.89 ± 6.02$$

HFDF 182.1 ± 5.432 248.75 ± 3.67 308. 97 ± 4.31 60.22 ± 5.90 **

HFDT 172.0 ± 4.189 234.98 ± 7.01 342.89 ± 5.12 99.02 ± 6.18 **

HFDC 175. 01 ± 8.192 235.45 ± 6.93 337. 59 ± 6.12 102.14 ± 3.09 **

HFDTC 168.45 ± 7.182 225.32 ± 5.98 295.55 ± 9.02 70.23 ± 4.99 **

HFDO 165.21 ± 2.321 221.45 ± 5.84 285.64 ± 4.37 64.19 ± 5.12 **

All values were expressed as mean ± SEM for six rats in each group. * P < 0.05 as compared to HFD group. $$ P < 0.001 as compared to control group. ** P < 0.001 as compared to HFD group

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Haque MRafiul, Ansari HS. Anti-Obesity Effect of Arq … Drug Res 2018; 68: 637–647

Original Article Thieme

(Pathogenic group). HFDT groups were significantly (p < 0.001) low-ered the increase levels of TC and LDL-cholesterol, but did not show significant reduction of enhanced TG. HFDC groups showed signif-icant (p < 0.001) reduction of raised TG level, but did not show any significant reduction in increase TC and LDL-cholesterol level. But both group HFDT and HFDC significantly raised the levels of re-duced concentration of HDL- cholesterol in plasma. It is shown in ▶table 4.

Theeffectofformulationarqzeeraanditsmaincomponentsonleptin,insulin,pancreaticlipase,andbloodglucose.Plasma concentration of glucose leptin, insulin levels and pancre-atic lipase activity in HFD group were significantly (p < 0.001) in-creased as compared to normal healthy group. HFDF, HFDC, HFDTC and HFDO were significantly (p < 0.001) reduced the serum increase concentration of glucose, Leptin and insulin levels while these groups significantly (p < 0.001) increased the reduced pancreatic lipase inhibition. HFDT groups were showed highly significant (P < 0.001) reduction of raised concentration of leptin in plasma but did not show any significant reduction of enhanced level of in-sulin, glucose level and, pancreatic lipase activity in blood plasma, but HFDC groups significantly (p < 0.001) lowered the levels of glu-cose and insulin in blood plasma but considerably decreased the raised pancreatic lipase activity. And HFDC group did not show sig-nificantly reduction of raised plasma level of leptin when compared to HFD groups. It is shown in ▶table 5.

TheeffectofformulationarqzeeraanditsmaincomponentsonliverenzymeSignificant (p < 0.001) elevated level of AST and ALT in HFD groups was found when compared with normal control groups. HFDF and HFDTC were significantly (p < 0.001) lowered the increased level of AST and ALT. And HFDO were showed significant (p < 0.001) reduc-tion of increased ALT level, but did not alter in increased AST level. HFDC were showed significant (p < 0.05) reduction of increased ALT level, but did not alter in increased AST level. HFDT were showed considerable reduction ((p < 0.05) of increase level of AST and ALT in the blood plasma shown in ▶Fig. 3 and 4.

TheeffectofformulationarqzeeraanditsmaincomponentsonlivertissueHistopathological study of liver showed the pronounce changes in Kupffer cell, portal Triad, hepatocytes vacuolation, sinusoidal dila-tion and lipofuchsin i. e., hepatic necrosis in HFD groups as com-pared to basal group. HFDF, HFDT, HFDC and HFDTC showed were significant protective effect as compared to HFD group. HFDO ex-hibited less ameliorative effect as compared to HFDF, HFDT, HFDC and HFDC shown in ▶Fig. 5

DiscussionA model of diet-induced obesity in rats is well controlled and shares many features with human obesity. Dietary fat is one of the most important environmental factors associated with the incidence of obesity which characterized by hyperphagic, hyperlipidemia, hy-perglycaemia, and hyperleptinemia [29–31]. A rodent model of

▶table 3 The results of the effect of formulation and its components on food intake, and visceral fat pat and liver weight.

Groups Food intake (g) Visceral fat pad weights (g) Liver weight (g)

Control 14. 13 ± 0.26 8.5 ± 1.18 4.96 ± 0.13

HFD 20.20 ± 0.23$$ 22.42 ± 4.2$$ 11.47 ± 0.42$$

HFDF 17.86 ± 0.384 ** 9.725 ± 2.22 ** 5.08 ± 0.138 **

HFDT 18.8 ± 0.489 * 17.10 ± 3.11 ** 9.02 ± .18 **

HFDC 19. 91 ± 0.492 18. 35 ± 1.22 ** 10.0 3 ± 0.24 **

HFDTC 18.38 ± 0.312 * 12.24 ± 3.12 ** 6.32 ± 1.01 **

HFDO 19.34 ± 0.248 8.69 ± 1.26 ** 5.23 ± 0.084 **

All values were expressed as mean ± SEM for six rats in each group. * P < 0.05 as compared to HFD group. $$ P < 0.001 as compared to control group. ** P < 0.001 as compared to HFD group

▶table 4 The results of the effect of formulation and its components on lipid profile.

Groups total triglycerides level (mg/dl)

total cholesterol level (mg/dl)

HDL-cholesterol level (mg/dl/)

LDL-cholesterol level (mg/dl)

Control 48.60 ± 2.51 62.82 ± 2.18 34.10 ± 0.89 18.956 ± 0.81

HFD 109.105 ± 4.37$$ 98.59 ± 2.80$$ 25.07 ± 2.59$$ 54.899 ± 3.81$$

HFDF 52.84 ± 1.89 ** 64.82 ± 1.39 ** 37.11 ± 1.60 ** 17.132 ± 0.80 **

HFDT 98.10 ± 3.82 76.0 ± 3.102 ** 33.36 ± 2.18 ** 23.22 ± 5.08 **

HFDC 85. 21 ± 7.54 ** 91. 81 ± 6.54 32. 35 ± 3.12 ** 42.61 ± 3.09

HFDTC 61.09 ± 3.11 ** 70.34 ± 2.09 ** 36.95 ± 1.43 ** 21.172 ± 2.19 **

HFDO 42.23 ± 3.361 ** 74.098 ± 1.174$$ 29.65 6 ± 1.09 ** 35.9 ± 1.81 **

All values were expressed as mean ± SEM for six rats in each group. * P < 0.05 as compared to HFD group$$ P < 0.001 as compared to control group. ** P < 0.001 as compared to HFD group

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Haque MR, Ansari HS. Anti-Obesity Effect of Arq … Drug Res 2018; 68: 637–647

obesity based on the intake of HFD is advantageous in studying obesity-related factors [32]. In present investigation, it was found that arq zeera and its main components could remarkable amelio-rated the increases in body weight, food intake, visceral fat pad, plasma lipid profile, AST, ALT, insulin, glucose, leptin and pancre-atic lipase activity and enhanced the decrease level of HDL-c dur-ing obese state. Especially, the effect of thymol + cuminaldehyde

on obese rats was better than that of thymol or cuminaldehyde alone, which indicates that thymol and cuminaldehyde might have a synergetic effect on obese rats. Although thymol is the main com-ponent of ajwain oil and cuminaldehyde is the main component of cumin and caraway oil, arq zeera contains many other effective components alph-terpinene, 1-phenyl propanol, 4-ethyl-3-nonen-5-yne, carvacrol, nerol, limonine, para cymene, 4- terpineol, zingi-berine etc. [33–35]. Therefore the antiobesity action mechanism of arq zeera may be intricate. It has been investigated that cumin oil containing cumic aldehyde, alpha-terpinene, 1-phenyl propanol, 4-ethyl-3-nonen-5-yne etc has antihyperlipidemia, and antihyper-glycaemia [34]. It is reported that alpha –terpinene has antihyper-lipidemia action in Triton WR1339-treated rats [36]. It is investi-gated that thymol has good antiobesity action in HFD induced mu-rine model [37]. Therefore in the current study, the preliminary antiobesity mechanism of action of arq zeera was investigated on HFD induced-obesity in rat’s model. In the present study, body weight gain was higher in rats fed with HFD as compared to the nor-mal healthy control rats. Matsuo et al., 2002 reported that body weight gain was greater in beef tallow diet group than in the other dietary groups [38]. Results from the present study showed that HFD induced obese rats a significant increase in food intake as well as visceral fat pad as compared to normal healthy control rats. The visceral fat pads (i. e. mesenteric, perirenal and epididymal fat) of HFD rats weighed 50 % more than those of normal healthy control rats as feeding of HFD in rats increases body weight, adiposity and visceral fat deposition [39]. Therapy of arq zeera and its main com-ponents to obese rats, significantly reduced the raised body, vis-ceral fat pad weight and food intake. The effect of thymol + cumi-naldehyde on body, visceral fat pad weight and food intake was found more significant than that of thymol or cuminaldehyde alone. We found that HFD for a period of 6 weeks produced significant in-crease in the serum TC, TGs and LDL-C levels in HFD-induced obe-sity in rats. Serum HDL-C levels were decreased in the HFD group rats as compared to the normal healthy control group rats. Our study results are supported by the work of Lavie and Milani, 2002, which indicated that obesity adversely, affects plasma lipids, espe-cially by increasing TC and decreasing the levels of HDL-C [40]. The HFD might lead to an increase in the synthesis of phospholipids and cholesterol esters in rats [41]. Generally, all lipids are absorbed into the blood in the gastrointestinal tract through a form of chylomi-crons, composed of triglycerides, phospholipids, cholesterol and apolipoprotein B. The triglycerides in these chylomicrons are then

▶table 5 The results of the effect of formulation and its components on insulin, leptin, glucose level and pancreatic lipase activity.

Groups Insulin level (µg/ml) Leptin level (ng/ml) Glucose level (mg/dl/ ) Pancreatic lipase activity (U/L)

Control 0.915 ± 0.07 1.14 ± 0.122 78.19 ± 1.361 165.03 ± 13.55

HFD 1.87 ± 0.087$$ 5.04 ± 0.2593$$ 131.92 ± 1.38$$ 558.17 ± 36.02$$

HFDF 0.95 ± 0.0482 ** 1.80 ± 0.1036 ** 80.64 ± 7.19 ** 180.35 ± 5.54 **

HFDT 1.60 ± 0.09 2.93 ± 0.11 ** 124.76 ± 4.22 498.02 ± 7.18

HFDC 1. 21 ± 0.192 ** 4.45 ± 0.93 103. 25 ± 4.32 ** 315.99 ± 3.19 **

HFDTC 1.03 ± 0.483 ** 2.10 ± 0.93 ** 86.34 ± 3.12 ** 210.21 ± 3.19 **

HFDO 1.02 ± 0.089 ** 2.53 ± 0.253 ** 111.178 ± 1.731 ** 141.46 ± 4.58 **

All values were expressed as mean ± SEM for six rats in each group. * P < 0.05 as compared to HFD group. $$ P < 0.001 as compared to control group. ** P < 0.001 as compared to HFD group

▶Fig. 3 The results of the effect of formulation and its components on AST enzyme. (All values were expressed as mean ± SEM for six rats in each group. * P < 0.05 as compared to HFD group. $$ p < 0.001 as compared to control group. ** p < 0.001 as compared to HFD group)

▶Fig. 4 The results of the effect of formulation and its components on ALT enzyme. (All values were expressed as mean ± SEM for six rats in each group. * P < 0.05 as compared to HFD group$$ p < 0.001 as compared to control group. ** p < 0.001 as compared to HFD group

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Haque MRafiul, Ansari HS. Anti-Obesity Effect of Arq … Drug Res 2018; 68: 637–647

Original Article Thieme

digested as fatty acids and glycerol by lipoprotein lipase. These fatty acids are transported and stored in liver and adipose tissues in the form of triglycerides. The remnants of the chylomicrons are mainly taken up by liver, and then transformed into several lipopro-teins containing triglycerides, phospholipids, cholesterol and apoli-poproteins. Among the lipoproteins, very low-density lipoproteins,

composed of triglycerides, cholesterol and phospholipids, trans-port triglycerides that are synthesized in the liver to adipose tis-sues. Its transforming, low-density lipoproteins (LDL), which con-sists of cholesterol and phospholipids, transport cholesterols into peripheral tissues [2]. As a result, increased intake of lipids in foods causes not only an accumulation of body fat, but also increases in

control HFD

HFDTHFDF

HFDC

HFDTC

HFDO

▶Fig. 5 Observation of Histopathological changes of liver in different groups (CV = Central Vein, KC = Kupffer cells,,Pig = lipofuscin pigment, Vac = vacuolated hepatocytes, PV = portal vein.

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Haque MR, Ansari HS. Anti-Obesity Effect of Arq … Drug Res 2018; 68: 637–647

cholesterols, phospholipids, and free fatty acids in the bloodstream that are transported to peripheral tissues as described. Also, excess carbohydrates and proteins in the body are converted to triglycer-ides, which are the main fats stored in adipose and liver tissues [2]. All of these cause obesity and obesity-derived cardiovascular dis-eases, such as hyperlipidemia and atherosclerosis. Excess lipid ac-cumulation is closely associated with insulin resistance, impaired glucose tolerance, and elevated serum TG concentration in obese subjects [42]. Arq zeera and thymol significantly lowered the raised total cholesterol in HFD -induced obese rats while cuminaldehyde did not any show significant effect on total cholesterol. But, inter-estingly thymol + cuminaldehyde therapy significantly reduced the increased levels of total cholesterol on obese rats. A substan-tial reduction of total cholesterol in serum by arq zeera, thymol and thymol + cuminaldehyde could be attributed to a reduction in the activities of the liver enzyme 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) reductase, which is a rate-limiting enzyme in choles-terol biosynthesis [43]. Arq zeera and its main components remark-able ameliorated the raised LDL-c and enhanced the decreased lev-els of HDL-c in obese rats. Also, a substantial reduction in LDL-cho-lesterol and total cholesterol level in serum could be achieved by decreased production of total cholesterol by liver tissue and/or ef-ficient removal of the LDL-cholesterol by various tissues without subsequent renewal. Significant elevated levels of total triglycer-ides in obese rats was remarkable ameliorated by the administra-tion of arq zeera, cuminaldehyde and thymol + cuminaldehyde but thymol did not significantly reduced the raised level of total triglyc-erides. Reductions in triglycerides in serum could be attributed to the inhibition of lipid absorption in the gastrointestinal tract, such as gastrointestinal lipase inhibition [44]. Consumption of HFD for a period of 6 weeks produced significant increase in the serum In-sulin, leptin and pancreatic lipase activity in HFD-induced obese rats. Insulin resistance is coupled with hypertriglyceridemia, low HDL-C and an enhanced secretion of LDL, as well as disorders of co-agulation and weight gain, and since obesity is a risk factor for dia-betes, insulin resistance predisposes to obesity and the develop-ment of diabetes. Insulin resistance is a state in which a given con-centration of insulin is associated with a subnormal glucose response [45]. However, over time, functional defects in insulin se-cretion prevent the ß-cells from maintaining high rates of insulin secretion, resulting in impaired glucose tolerance and eventually diabetes mellitus type 2, the deregulation of carbohydrate and lipid metabolism that occurs as a consequence of insulin resistance ex-acerbates the progression of insulin resistance, this leads to the de-terioration of glucose homeostasis and the development of glu-cose intolerance, which after a while leads to diabetes, and adipose cells produce more fatty acids, whereas the liver generates more glucose in an up regulated manner, and the ß-cells undergo com-plete failure resulting in the late stages of diabetes which needs high doses of exogenous insulin. Administration of the formulation arq zeera, cuminaldehyde and thymol + cuminaldehyde consider-ably decreased the raised concentration of insulin resistance, result in reduced the impaired glucose tolerance, but thymol did not show any considerable effect. Thus arq zeera can also be used for the treatment of diabetes mellitus type 2 HFD induced obese rats. Lep-tin alters the release of several neuropeptides after binding to its receptors (leptin receptor (LepR), especially neuropeptide Y (NYP)

from the hypothalamus and in the hypothalamus leptin inhibits the expression of NPY [46]. Neuropeptide Y acts as a transmitter in the nervous system which is important due to its appetite stimulating effect. High hypothalamic concentrations of neuropeptide Y elicit food intake [47]. Arq zeera, thymol and thymol + cuminaldehyde-lo wered the increased plasma leptin level, result in elicits the food intake and enhanced energy expenditure while cuminaldehyde but cuminaldehyde did not significantly lowered the elevated plasma leptin level in obese rats. Pancreatic lipase is the key enzyme for lipid absorption that hydrolysis triglycerides in the gastrointestinal tract. Pancreatic lipase inhibitor which helps to limit intestinal fat absorption at the initial stage, have been proved as useful medica-tions for the treatment of hyperlipidemia and a great promise as antiobesity agents [48]. Arq zeera and its main components sig-nificantly inhibit the pancreatic lipase activity on obese rats and thus it prevents lipid absorption that hydrolysis triglycerides in the gastrointestinal tract. Liver steatosis is a well-known pathology in severely obese patients and is especially associated with visceral adiposity and diabetes. It may progress in some patients to steato-hepatitis and cryptogenic cirrhosis [49]. In many of obese people, increase of hepatic triglyceride levels, causes hepatic steatosis [50]. Arq zeera and its main components treatment decreased lipogen-ic ACC expression in epididymal adipose tissue of HFD induced obese rat and protect to liver steatosis by regulate the hepatic tri-glyceride levels and excess pronounce changes in Kupffer cell, por-tal Triad, hepatocytes vacuolation, sinusoidal dilation and lipofuch-sin in the hepatic cell. The elevated levels of liver enzyme (AST and ALT) and hepatic fat deposition in obese rat were lowered consid-erably by therapy of arq zeera and thymol + cumic aldehyde. Thy-mol was significantly reduced the increased levels AST and ALT in obese rats, while, cuminaldehyde significantly reduced the in-creased ALT levels and did not alter the increased AST level in obese rats. In comparison to synthetic orlistat antiobesity drug, herbal preparation arq zeera showed more significant reduction of raised body, food intake, visceral fat pad weight and total plasma choles-terol, LDL-c, glucose, insulin and leptin levels and enhanced the lowered level of HDL-c in obese rats while orlistat showed more pancreatic lipase inhibition and decreased the raised level of total triglycerides than arq zeera and its components on obese rats. Thus, over all of the results showed that arq zeera and thy-mol + cuminaldehyde had a potential antiobesity effect in HFD in-duced obese rat model. Our results also suggest that the tradition-al herbal preparations have good quality, therapeutics action and without any risk of significant adverse effect as compared synthet-ic medicine.

ConclusionsIn conclusion, according to our presents finding, the arq zeera and its main components (thymol and cumic aldehyde) possess potent antiobesity effect on HFD-induced obese rats. Thymol therapy ex-erted antiobesity effect by significantly lowered the increased food intake, total cholesterol and leptin level while cuminaldehyde ex-erted antiobesity effect by significantly reduced the increased of total triglycerides, glucose, insulin level and pancreatic lipase ac-tivity in obese rats. Moreover thymol and cuminaldehyde had a syn-ergistic effect on obese rats and it demonstrated that the compat-

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Haque MRafiul, Ansari HS. Anti-Obesity Effect of Arq … Drug Res 2018; 68: 637–647

Original Article Thieme

ibility of ajwain, cumin and caraway was reasonable in the treat-ment of obesity. Therapy of arq zeera had more significant antiobesity effect than thymol + cuminaldehyde therapy and it clearly indicate that the synergist effect of four herbs, ajwain, cumin, ginger and caraway was reasonable in the treatment of obe-sity. The antiobesity effect of arq zeera was found to be more ef-fectual than orlistat. These finding strengthen the observation that polyherbal formulation or naturally occurring compounds of plant origin are much more effective in controlling obesity without show-ing any significant adverse effect than synthetic oral antiobesity drugs.

AcknowledgementThe present research was supported by a grant from CCRUM (dept of AYUSH, Ministry of Health & Family Welfare, Government of India). Authors are thankful to Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy for providing all necessary facilities for this work.

Conflict of Interest

The authors declare no conflict of interest.

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