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1 Article Title Luffa cylindrica: a promising herbal treatment in progesterone induced obesity in mice 2 Article Sub- Title 3 Article Copyright - Springer Nature Switzerland AG 2021 Year (This will be the copyright line in the final PDF) 4 Journal Name Journal of Diabetes & Metabolic Disorders 5 Family Name Biradar 6 Particle 7 Given Name Prakash Rajshekhar 8 Suffix 9 Corresponding Organization KLE Academy of Higher Education and Research Author (KAHER) 10 Division Department of Pharmacology and Toxicology, KLE College of Pharmacy, Belagavi 11 Address Belagavi 590010, India 12 e-mail [email protected] 13 Family Name Wilnellia 14 Particle 15 Given Name D’silva Wilnoska 16 Suffix

17 Author Organization KLE Academy of Higher Education and Research (KAHER) 18 Division Department of Pharmacology and Toxicology, KLE College of Pharmacy, Belagavi 19 Address Belagavi 590010, India 20 e-mail 21 Author Family Name Patil 22 Particle 23 Given Name Ashiwini 24 Suffix 25 Organization KLE Academy of Higher Education and Research (KAHER) 26 Division Department of Pharmacology and Toxicology, KLE College of Pharmacy, Belagavi 27 Address Belagavi 590010, India 28 e-mail 29 Received 31 August 2020 30 Schedule Revised 31 Accepted 20 January 2021 32 Abstract Objective: The present study investigates the anti-obesity activity of Luffa cylindrica in progesterone induced obesity model. Method: Swiss albino mice were grouped into 7 (n = 6). Obesity was induced by administration of progesterone (10 mg/kg s.c.) for 28 days.Normal group received ad libitum and water. Disease control, positive control and treatment groups received progesterone (10 mg/kgs.c.). Further positive control group received Orlistat (10 mg/kg p.o). Treatment groups received hydro-alcoholic and fractionation extract of Luffa cylindrica at doses 400, 200, 100 mg/kg b.w. Food and water intake were recorded daily, Body weight, BMI and blood glucose were checked weekly. On completion of the study, animals were sacrificed and blood serum being utilized to accesses in vitro the estimate the various biochemical parameters. Serotonin levels and antioxidant biomarkers were also estimated. Histopathology of liver and adipose tissue was studied. Result: After the 28 days of treatment with plant extract and fraction, it was observed to improve the progestrogen-induced obesity by improving BMI, body weight, brain serotionin, locomotor activity, blood glucose level, anti- oxidant biomarkers, and lipid profile. Conclusion: After assimilating the In vivo and in vitro studies Luffa cylindrical extract highlighted the antilipidemic, anti-hyperglycaemic, antioxidant potential in hormone-induced obesity. 33 Keywords separated Luffa cylindrica - Obesity - Progesterone - Serotonin - Orlistat by ' - ' 34 Foot note Springer Nature remains neutral with regard to jurisdictional claims in information published maps and institutional affiliations. JrnlID 40200_ArtID 749_Proof# 1 - 31/01/2021

Journal of Diabetes & Metabolic Disorders https://doi.org/10.1007/s40200-021-00749-8 1 34 RESEARCH ARTICLE 2

5 6 Luffa cylindrica: a promising herbal treatment in progesterone 7 induced obesity in mice

8 D’silva Wilnoska Wilnellia1 & Prakash Rajshekhar Biradar 1 & Ashiwini Patil1 9

10 Received: 31 August 2020 /Accepted: 20 January 2021 11 # Springer Nature Switzerland AG 2021

12 Abstract 13 Objective The present study investigates the anti-obesity activity of Luffa cylindrica in progesterone induced obesity model. 14 Method Swiss albino mice were grouped into 7 (n = 6). Obesity was induced by administrationOF of progesterone (10 mg/kg s.c.) 15 for 28 days.Normal group received ad libitum and water. Disease control, positive control and treatment groups received 16 progesterone (10 mg/kgs.c.). Further positive control group received Orlistat (10 mg/kg p.o). Treatment groups received 17 hydro-alcoholic and fractionation extract of Luffa cylindrica at doses 400, 200, 100 mg/kg b.w. Food and water intake were 18 recorded daily, Body weight, BMI and blood glucose were checked weekly. On completion of the study, animals were sacrificed 19 and blood serum being utilized to accesses in vitro the estimate the variousPRO biochemical parameters. Serotonin levels and 20 antioxidant biomarkers were also estimated. Histopathology of liver and adipose tissue was studied. 21 Result After the 28 days of treatment with plant extract and fraction, itD was observed to improve the progestrogen-induced 22 obesity by improving BMI, body weight, brain serotionin, locomotorE activity, blood glucose level, anti-oxidant biomarkers, and 23 lipid profile. 24 Conclusion After assimilating the In vivo and in vitro studies Luffa cylindrical extract highlighted the antilipidemic, anti- 25 hyperglycaemic, antioxidant potential in hormone-induced obesity.

26 Keywords Luffa cylindrica . Obesity . Progesterone . Serotonin . Orlistat 27

28 ORRECT 29 Introduction C prevalence reports adjusted for age, at least 30% of males and 40 35% of females were noted to be obese [3, 4]. 41 30 Obesity occurs as a result of a disparity between energy ex- BMI, which is a value derived by calculating the ratio of 42 31 penditure and energy intakeUN which leads to an increase in the body weight in kilograms to the square of the height in 43 32 circulatory lipid concentration and enlargement of fat mass. meters, is used to define normal weight, overweight and obe- 44 33 The aetiological factors of obesity include fluctuations in the sity. Normal: BMI 18.5-24.9 kg/m2, overweight: BMI 25-29.9 45 34 circulatory levels of certain hormones, such as leptin, insulin, kg/m2, class I obese: BMI 30-34.9 kg/m2, class II obese: BMI 46 35 sex hormones and growth hormones that majorly influence 35-39.9 kg/m2, and class III obese: BMI 40 or greater kg/m2 47 36 appetite, body fat distribution, metabolism, storage and also [morbidly obese] [5, 6]. 48 37 influenced by various lifestyle factors [1, 2]. The ubiquity of Obesity is a common risk factor for multiple comorbidity 49 38 obesity is noted to have increased multifold in the preceding conditions such as cardiovascular diseases, hypertension, dys- 50 39 few decades. According to a recent estimate of global obesity lipidemia, type-2 diabetes mellitus and insulin resistance. The 51 prevalence of these comorbidities is directly proportionate to 52 the degree of obesity [7–9]. 53 54 * Prakash Rajshekhar Biradar Amongst the hormones that modulate food intake and en- [email protected]; [email protected] ergy balance in mammals, the sex hormones estrogen and 55 progesterone are considered to have a prominent role [10]. 56 Progesterone is produced endogenously and has the primary 57 1 Department of Pharmacology and Toxicology, KLE College of 58 Pharmacy, Belagavi, KLE Academy of Higher Education and role of regulating embryogenesis, pregnancy, lactation and Q1 Research (KAHER), Belagavi 590010, India menstrual cycle [11]. The use of progesterone containing 59 JrnlID 40200_ArtID 749_Proof# 1 - 31/01/2021

J Diabetes Metab Disord

60 preparations in contraceptives and hormone replacement ther- Extraction of plant material 108 61 apy has been shown to cause hyperphagia and increased fat 62 deposition leading to substantial weight gain [12, 13]. .Some 200 gms of coarsely powdered plant material was macerated 109 63 women have been noted to develop a depressive illness due to with hydro-alcohol for seven days in the ratio of 7:3with oc- 110 64 an increase in progesterone secretion [14, 15]. Progesterone casional shaking. The macerated solute was filtered using 111 65 has been proven to induce hyperphagia by modulating sero- Whatman filter paper and the filtrate stored. With the aim of 112 66 tonergic agents [16, 17]. doing Soxhlet extraction, 100 g of marc was taken in 400 ml 113 67 Obesity has been considered to be a chronic disease of ethanol for a period of 72 hours at 40°C (flask size 400 ml). 114 68 and hence anti-obesity medications are prescribed for The solution thus obtained was then filtered. Following this, 115 69 long-term treatment which have numerous side effects the two filtrates, ie the one obtained post maceration and the 116 70 [18]. There are several herbal plants whose active compo- one obtained post Soxhlet extraction, were mixed and a con- 117 71 nents are being used in anti-obesity treatment, given their centrate prepared in the rotary evaporator. The resultant con- 118 72 safety, efficacy, cultural acceptability and a more accept- centrate was then transferred onto a Petri plate and evaporated 119 73 able side-effect profile [19, 20]. It has been reported that on a water bath. The final filtrate obtained post these proce- 120 74 movement behavior profiles across work and leisure time- dures, was stored in an air tight container and lyophilized to 121 75 use compositions are associated with obesity [21] powered extract. 122 76 reflecting the requirement of adequate balance between OF 77 work and leisure movement. Fractionation 123 78 Luffa cylindrica or Luffa aegyptiaca is a member of the 79 Cucurbitaceae family. Used over generations as an herbal Using a separating funnel, a crude extract was prepared by 124 80 remedy in different countries for the treatment of various types mixing dichloromethanePRO and citric acid 5% thoroughly. The 125 81 of diseases. The plant contains a group of phytoconstituents to aqueous layer obtained was separated and concentrated on a 126 82 include alkaloids, phenols, flavonoids, saponins, tannins, ste- Petri plateD and ammonia was added to make up the pH to 9.2. 127 83 roids, ascorbic acid, triterpenoids, and oleanolic acid render- TheE residue was again extracted with dichloromethane and 128 84 ing it an efficacious medicinal plant. Most studies have report- citric acid in the ratio of 1:1. The citric acid layer was concen- 129 85 ed that these bioactive compounds have promising effects in trated to give flavonoids and saponins. Finally, the mixture 130 86 tackling obesity by several mechanisms [22, 23]. was extracted again with 90% methanol and acetone to obtain 131 87 The saponins isolated from the aerial parts of Luffa a precipitate which contained saponins [25]. 132 88 cylindrica have traditional claim to possess anti-obesity prop- 89 erties, its role in the management of obesity [24]. No study had Experimental animals 133 90 been done in relation to its Anti-obesity effects. Thus the cur- 91 rent study is designed to evaluate the anti-obesity properties of The Institutional Animal Ethics Committee, KLEU’s College 134 92 Luffa cylindrica in female Swiss albino miceORRECT under laboratory of Pharmacy, Belgaum (IAEC Reg. No.221/Po/Re/S/2000/ 135 93 conditions. C CPCSEA) reviewed and approved the study. Healthy Swiss 136 albino female mice weighing between 20 to 25 gm. were 137 chosen as animal experimental models for the study. 138 UN Specimen mice were procured from In Vivo Biosciences, 139 94 Material and methods Bangalore authenticated animal breeder. The mice were ac- 140 commodated in a clean and transparent polypropylene cage in 141 95 Collection of plant materials groups of six subjects per cage, maintained under 12/12 hr. 142 natural light-dark cycle at 25±2°C ambient temperature, 45-55 143 96 A fresh sample of locally growing Luffa cylindrica be- % relative humidity for 7 days for acclimatized . The mice 144 97 longing to family Cucurbitaceae was sourced from were allowed free access to standard pellets and water. 145 98 Betalbatim in Goa in August. Post being identified and 99 authenticated by Dr. Harsha Hegde, Scientist D, ICMR- Experimental groups 146Q2 100 National Institute of Traditional Medicine, Belagavi- 101 Karnataka, it was Authenticated and the voucher No. Group I (normal): Normal group 147 102 RMRC-1556 of the herbarium. Processing of the plant. Group II (disease control): Receives progesterone 148 103 After procurement, the collected material was shade dried (10 mg/kg) s.c. 149 104 and rendered 100% free of moisture. A coarse powdery Group III (positive control): Receives progesterone 150 105 residue of it was then prepared by first chopping the dry (10 mg/kg) s.c. + Orlistat at dose of (10 mg/kg) p.o. 151 106 and moisture free remnants of the shade-dried plant ma- Group IV (treatment group): Receives progesterone 152 107 terial and then powdered the same. (10 mg/kg) s.c. + LC 400 mg/kg p.o. 153 JrnlID 40200_ArtID 749_Proof# 1 - 31/01/2021

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154 Group V (treatment group): Receives progesterone was administered subcutaneously (10mg/kg) in the dorsal 196 155 (10 mg/kg) s.c. + LC 200 mg/kg p.o. neck region. Group III was administered Orlistat at dose of 197 156 Group VI (treatment group): Receives progesterone (10 mg/kg) Group IV, V and VI received Luffa cylindrica 198 157 (10 mg/kg) s.c. + LC 100 mg/kg p.o. extract at dose 400, 200, 100 mg/kg bw respectively and 199 158 Group VII (treatment group): Receives progesterone Group VII received fractionation of Luffa cylindrica extract 200 159 (10 mg/kg) s.c. + Fractionation of LC 200 mg/kg p.o. at dose of 200mg/kg bw. After 30 mins Group II, III, IV, V, VI 201 , VII received progesterone (10mg/kg) subcutaneously in the 202 dorsal neck region for 28 days. 203 160 Induction of obesity Body weight and BMI 204 161 The selected mice models were injected with progesterone for 162 28 days via the subcutaneous route at the dorsal neck region to The bodyweight of the mice (in gm.) and length (nasal to anal 205 163 induce obesity. The dose used for induction was measured at length in cm) was recorded on 0th,7th,14th 21st an 28th day just 206 164 10 mg/kg bw and was prepared by using Arachis oil as a before the administration of test drugs. This was done using 207 165 medium for dissolving the progesterone for injecting pur- the precision balance with 10 mg sensitivity and a scale. To 208 166 poses. The drug extract was administered to all of the exper- determine body mass index of mice, Lee index was used, 209 167 imental models 30 minutes prior to the administration of pro- which was defined as OF 210 168 gesterone [26]. pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 3 body weight BMI ¼ 1000 169 Preparation of standard drug solution length from nose to anus PRO 213 Mice with BMI ≥ 310 were considered obese [27]. 211212214 170 As Orlistat is soluble in water, a solution of the drug in dis- 171 tilled water was prepared to be used as the standard drug D 215 172 solution. The volume of the dose administered was10mg/kg ExploratoryE behaviour 173 bw. For the entire duration of the study period, every week, thirty 216 174 Dose selection minutes after undertaking test drug administration to treatment 217 groups, the exploratory behaviour of the mice in each group 218 219 175 Acute toxicity studies: The volume of the dose to be admin- was recorded. The mice were allowed to acclimatize to labo- 220 176 istered was selected as per the reported LD50 value. Adhering ratory conditions for one hour before the exploratory behavior 221 177 to OECD guidelines no-423, acute toxicity studies were car- was recorded. The recording procedure began by placing the 222 178 ried out, and a dose of 2000 mg/kg bw was reported to be safe mice (one group at a time) in the centre of a field consisting of 223 179 in mice Therefore 1/5th,1/10thand1/20thdoseORRECT of hydroalcoholic 25 squares (8 cm×8 cm) surrounded by a wall thickness of 15 224 180 extract of Luffa cylindrica were taken for the study. mm. A record of their open field activity was made by noting C 225 181 Fractionation of saponins dose 200m/kg bw was selected. the number of squares they crossed in horizontal movement, 226 182 Route of administration: The oral route was selected for the how frequently they tried to rear, ie stand up vertically, for five 227 183 administration of the drug. UN minutes and the frequency of grooming (face wiping).

184 Preparation of test solution Determination of blood glucose level 228

185 As the Luffa cylindrica extract is water-soluble, hence distilled Blood samples were collected from the caudal vein of the 229 th th th st th 186 water was chosen as the selected medium to dissolve the ex- mice on day 0 ,7 ,14 ,21 and 28 . An estimation of their 230 187 tract and prepare the test solution. All the drug of different blood glucose levels was performed with the help of a digital 231 188 concentrations to be used during the study were prepared glucometer and glucose strips. The readings obtained were 232 189 freshly just before administration. The test drug and the stan- recorded in mg/dl. 233 190 dard drug were administered to the animal subjects via oral 191 gavage. Biochemical estimation 234

192 Anti-obesity assay Preparation of serum 235

193 Female swiss albino mice were divided into seven groups of Following the last test drug administration, on Day 29th of the 236 194 six mice each and treated as follows: Group I (Normal group) study, anesthetized with diethyl ether. Then making a punc- 237 195 no treatment given, Group II (disease control) progesterone ture retro-orbital, blood for serum preparation was extracted 238 JrnlID 40200_ArtID 749_Proof# 1 - 31/01/2021

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239 and collected in sterilized, single-use Eppendorf tubes using Histopathological study 283 240 heparinized glass capillary. To aspirate the plasma, the blood 241 sample was then centrifuged for 10 minutes at a speed of 3000 At the end of the study, animals were sacrificed and their liver 284 242 rpm. The plasma thus derived was used to prepare a profile of and adipose tissue were isolated and kept in 10% formalin. 285 243 the blood lipids of the subjects. The serum to be analysed was Sections of the tissues fixed in paraffin were prepared stained 286 244 stored in the refrigerator. with hematoxylin and eosin and observed for pathological 287 changes. 288

245 Lipid profiles Statistical analysis 289

246 All analyses of serum were completed within 24h of sample Results were expressed as mean ±SEM. One way ANOVA 290 247 collection Autoanalyser was used to analyse lipid profile was utilized to determine the differences in the data followed 291 248 Triglycerides (TG) Total cholesterol (TC) High density lipo- by Dunnett Multiple Comparison Test (Graph pad Prism soft- 292 249 protein (HDL) using ERBA kits Whereas the low density ware, version 8.3.1). A reading of p˂0.05 was considered 293 250 lipoprotein (LDL) and very low density lipoprotein (VLDL) statistically significant. 294 Q3 251 levels were calculated according to Friedwald’sformula[28]. OF LDL cholesterolðÞ¼ mg=dl TC–ðÞHDL þ VLDL VLDL cholesterolðÞ¼ mg=dl TG=5 Results 295 254 252253 Effect of LuffaPRO cylindrica on body weight 296 255 Estimation of liver enzymes and blood pressure The variation in the body weight of the mice in different 297 groupsD was compared at the end of the study, i.e. on the 28th 298 256 Liver function test (SGPT and SGOT) was also carried out. day.E Initially there was a non-significant increase in body 299 257 All the biochemical parameters were carried out using ERBA weight, followed by a gradual increase on the 14th day in the 300 258 kit. Anti-oxidant enzymes SOD, GSH, CAT were analyser progesterone administered group when compared to that of 301 259 using autoanalyser [29]. The blood pressure was measured normal as seen in Table 1. There was a highly significant 302 260 using non-invasive blood pressure NIBP by tail cuff method increase (p˂0.0001) on the 21st and 28th day in the progester- 303 261 using MP100. one administered group as compared with the normal. 304 Treatment with Luffa cylindrica extract and fractionation of 305 saponins extract at all doses showed a significant reduction in 306 262 Estimation of serotonin in mice brain [30] body weight on the 14th day whereas on the 21st and 28th day 307 ORRECTthe body weight was highly significantly (p˂0.0001) reduced. 308 263 After taking the blood for serumC estimation dissection 264 was performed on each mice brain, separating the whole Effect of Luffa cylindrica on BMI 309 265 brain and the sub-cortical region. Homogenization of the 266 brain was done in 5 mlUN HCl-butanol solution for ap- The progesterone administered group showed significant 310 267 proximately1 min. Centrifugation of the ample was un- (p˂0.001) increase in BMI on the 14th and 21st days of the 311 268 der taken for 10min at2000r.p.m. Following this, 1ml study whereas on the 28th day BMI showed was a highly 312 269 aliquot of the supernatant phase was removed and trans- significant (p˂0.0001) increase when compared with the nor- 313 270 ferred into a centrifuge tube already containing 2.5 ml mal group. BMI of mice that were administered with doses of 314 271 heptane and 0.31 ml HCl of 0.1 ml. After shaking the the extract at 200 and 100 mg/kg bw and fractionation of 315 272 tube vigorously for 10 minutes, it was centrifuged fol- saponins extract at 200 mg/kg bw showed a similar trend with 316 273 lowingthesameprocedureasaboveandthetwophases that of the normal group, whereas when compared with the 317 274 were separated. The overlying organic phase was sepa- progesterone-administered group showed highly significant 318 275 rated and 0.2 ml of the aqueous phase was then utilized (p˂0.0001) decrease in BMI on the 21st and 28th day 319 276 for serotonin estimation. (Table 2). 320 277 Fluorophore was developed by heating a solution of 0.2ml 278 of aqueous extract to 0.25ml of OPT reagent at 100°C for Effect of Luffa cylindrica on exploratory behavior 321 279 10minutes. The sample was then allowed to reach an equilib- 280 rium at room temperature following which, spectrofluorome- A highly significant decrease in ambulation, rearing and 322 281 ter readings were taken at 360-470 nm. For the blank, 0.25 ml grooming (p˂0.0001) and (p˂0.001) was observed in the 323 282 of concentrated HCl was used without OPT reagent. progesterone-administered group when compared to the 324 JrnlID 40200_ArtID 749_Proof# 1 - 31/01/2021

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t1:1 Table 1 Effect of Various Treatments on Mice Body Weight

t1:2 Groups 0TH DAY 7TH DAY 14TH DAY 21ST DAY 28TH DAY

t1:3 Normal group 24.33 ± 1.43 25.01 ± 1.40 25.7 ± 1.302 26.15 ± 1.29 26.58 ± 1.27 t1:4 Disease group 24.01 ± 2.15 25.05 ± 1.91 28.53 ± 1.93** 29.93 ± 1.874*** 30.45 ± 1.74*** t1:5 Positivecontrol 23.51 ± 1.0 25.13 ± 0.92 25.38 ± 0.88## 24.08 ± 1.35### 23.81 ± 1.34### t1:6 LC 400 mg/kg bw 26.61 ± 0.72 28.08 ± 0.59 26.25 ± 0.6## 25 ± 0.41### 24.48 ± 0.33### t1:7 LC 200 mg/kg bw 26.31 ± 1.44 27.78 ± 1.42 25.61 ± 1.3## 24.93 ± 1.307### 23.88 ± 1.24### t1:8 LC 100 mg/kg bw 25.15 ± 0.96 26.86 ± 1.25 24.7 ± 0.982## 24.18 ± 1.12### 23.91 ± 1.12### t1:9 FRC LC 200 mg/kg bw 25.23 ± 0.88 27.51 ± 0.87 25.31 ± 1.4## 22.91 ± 0.821### 20.98 ± 0.72###

*p <0.05,**p < 0.001, ***p < 0.0001 when compared with NORMALGROUP # p <0.05,## p <0.001,### p <0.0001whencomparedwithDISEASE CONTROL

325 normal group, whereas mice treated with Luffa cylindrica ex- significant (p˂0.0001) reduction in blood sugar levels on the 345 326 tract 200 mg/kg bw and fractionation extract of saponins for 28th day (Table 4). OF 346 327 28days showed a highly significant (p˂0.0001) increase in 328 ambulation. Rearing and grooming also saw a significant in- 329 crease (p˂0.001) in the above-mentioned groups. Groups re- Effect of Luffa cylindrica on lipid profiles 347 330 ceiving Luffa cylindrica extract 400 and 100mg/kgbw showed PRO 331 a significant increase in ambulation, rearing and grooming in There was a highly significant increase (p˂0.0001) in the TG, 348 332 comparison to the progesterone-administered group (Table 3). TC, LDLD and VLDL levels and a highly significant decrease 349 (p˂E0.0001) in HDL levels in progesterone administered mice 350 when compared to normal group. The positive control group 351 333 Effect of Luffa cylindrica on blood glucose showed highly significant decrease in TG, TC, LDL and 352 VLDL levels whereas there was highly significant increase in 353 334 The blood sugar levels of the progesterone-administered mice the HDL levels where compared to progesterone administered 354 335 showed a highly significant increase (p˂0.0001) from the 7th groups. At dose 400 and 100 mg/kg bw of the extract showed 355 336 to the 28th day in comparison to the normal group. On the14th highly significant decrease in TG and VLDL levels further TC 356 337 day of treatment, there was no significant (p˂0.05) reduction levels were significantly decrease LDL levels showed a non- 357 338 in glucose level in all the treatment groups. However, on the significant change and significant decrease at 400 and 100 mg/kg 358 339 21st day, there was a significant (p˂0.001)ORRECT decrease in the respectively, whereas the HDL level showed a significant in- 359 340 blood glucose levels and the 28thC day of treatment with crease. Dose 200 mg/kg showed highly significant decrease in 360 341 Luffa cylindrica extract and fractionation of saponins extract the TG, TC, VLDL and LDL levels whereas the HDL levels 361 342 at all doses marked a highly significant (p˂0.0001) decrease in showed a highly significant increase. Fractionation of saponins 362 343 the blood glucose levels. TheUN treatment of obese mice with the extract exhibited a highly significant decrease in TG, TC, VLDL 363 344 standard drug Orlistat at a dose of 10 mg/kg showed a highly and LDL levels and highly significant increase in HDL levels 364

t2:1 Table 2 Effect of Various Treatment on Mice BMI

TH t2:2 GROUPS 0 DAY 7TH DAY 14TH DAY 21ST DAY 28TH DAY

t2:3 NORMAL GROUP 301.1333 ± 14.038 308.02 ± 1.0431 311.2 ± 0.937 311.2 ± 0.9370 309.8833 ± 6.0020 t2:4 DISEASE GROUP 309.4666 ± 7.9966 318.05 ± 3.3139 328.6083 ± 5.6662** 334.15 ± 5.4429** 336.325 ± 5.0443*** t2:5 POSITIVE CONTROL 309.0333 ± 6.4290 316.25 ± 6.0124 317.1166 ± 5.1938 311.35 ± 7.0142#### 309.8333 ± 6.9486### t2:6 LC 400 mg/kg bw 319.316 ± 2.3058 321.75 ± 2.8059 317.55 ± 2.2616 312.91 ± 2.2670## 310.7888 ± 2.4319## t2:7 LC 200 mg/kg bw 314.75 ± 2.0390 320.6716 ± 2.7502 312.1 ± 2.2217 309.2888 ± 2.2812### 304.7 ± 1.8873### t2:8 LC 100 mg/kg bw 313.2333 ± 2.1677 320 ± 1.8286 310.2666 ± 1.4354 308.9333 ± 1.4689### 307.6833 ± 1.3224### t2:9 FRC LC 200 mg/kg bw 316.1166 ± 1..9686 325.8333 ± 3.0943 312.4333 ± 0.6676 306.3166 ± 3.1069### 297.6833 ± 2.5799###

*p<0.05, **p<0.001, ***p<0.0001 when compared with NORMALGROUP # p<0.05, ## p<0.001, ### p<0.0001 when compared with DISEASE CONTROL JrnlID 40200_ArtID 749_Proof# 1 - 31/01/2021

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t3:1 Table 3 Effect of Various t3:2 Treatments on Exploratory Groups AMBULATION REARING GROOMING Behaviour t3:3 NORMAL GROUP 88.1666 ± 1.8693 20.5 ± 1.1180 5.3333 ± 0.9888 t3:4 DISEASE GROUP 51.1666 ± 2.3054*** 13.333 ± 1.1155** 2.5 ± 0.4281** t3:5 POSITIVE CONTROL 62.3333 ± 2.8480** 15.8333 ± 1.0775** 4 ± 0.3651 t3:6 LC 400 mg/kg bw 65.5 ± 2.4324## 17.6666 ± 0.6666## 3.5 ± 0.56273# t3:7 LC 200 mg/kg bw 71.3333 ± 4.5215### 18 ± 0.8563## 4.3 ± 0.6146## t3:8 LC 100 mg/kg bw 62.8333 ± 1.9220## 17.1666 ± 0.9457## 3 ± 0.4472## t3:9 FRC LC 200 mg/kg bw 79.5 ± 0.9574### 19.6666 ± 0.5577## 4.6666 ± 0.6146##

*p < 0.05, **p < 0.001, ***p < 0.0001 when Compared with NORMAL GROUP # p < 0.05, ## p < 0.001, ### p < 0.0001 when compared with DISEASE CONTROL

365 where compared to progesterone administered groups (Table 5). CATwhereasadoseofLuffa cylindrica extracts 200 387 366 Similarly, the result of SGOT and SGPT is shown in Table 6. mg/kg bw and FRC of saponins extract weighing 200 388 mg/kg body weight exhibited highly significant 389 (p˂0.0001) rise. Treatment of obese mice with Luffa 390 367 Effect of Luffa cylindrica on serotonin levels OF cylindrica extract at 400, 200, and 100 mg/kg bw and 391 FRC of saponins extract at 200 mg/kg bw demonstrated 392 368 The serotonin levels in the brain were highly significantly re- a highly significant (p˂0.0001) rise in SOD levels. Also, 393 369 duced (p˂0.0001) in the progesterone-treated group of mice treatment with the standard drug Orlistat at a dosage of 10 394 370 when compared with the normal group of mice with mean PRO mg/kg bw highly significantly increased (p˂0.0001) SOD, 395 371 valuesof0.38±0.55and0.82±0.22.Ontreatmentfor28days, there GSH, CAT levels in mice (Table 8). 396 372 showed a highly significant increase (p˂0.0001) in serotonin D 373 levels in groups receiving Luffa cylindrica extract measuring E 374 200mg/kgbw and fractionation of saponins extract measur- Effect of LC on blood pressure 397 375 ing 200mg/kgbw with mean values 0.67±0.45 and 0.78±0.22. 376 Groups receiving Luffa cylindrica extract at doses of 400 and Progesterone-administered mice showed a non-significant 398 377 100 mg/kg bw also showed a significant increase (p˂0.001) in impact on blood pressure on being compared with normal. 399 378 serotonin levels in the brain (Table 7). The administration of the extract exhibited a non- 400 significant decrease in blood pressure. This indicates that 401 402 379 Effect of Luffa cylindrica on liver anti-oxidant LC extract has no impact on the blood pressure levels in 403 380 enzymes ORRECTprogesterone-administered mice (Table 9).

381 The liver homogenate of experimentalC mice to which pro- Effect of Luffa cylindrica extract on histopathology of liver 404 382 gesterone was administered exhibited a highly significant and adipose tissue 405 383 decrease (p˂0.0001) in SOD,UN CAT and GSH levels when 384 compared with the normal group. Treatment of obese Histopathological studies of progesterone-administered mice 406 385 mice with Luffa cylindrica extract 400 and 100 mg/kg demonstrated pathological changes which include venous 407 386 bw resulted in a significant rise (p˂0.001) of GSH and congestion, sinusoidal congestion, Kupffer cell hyperplasia 408

Q4 t4:1 Table 4 Effect of various treatment on blood glucose

t4:2 GROUPS 0TH DAY 7TH DAY 14TH DAY 21ST DAY 28TH DAY

t4:3 NORMAL GROUP 49.1666 ± 0.9457 52.5 ± 1.0567 59.3333 ± 1.1737 68.166 ± 1.0137 82.166 ± 0.7490 t4:4 DISEASE GROUP 51.6666 ± 0.7149 83.3333 ± 0.8027*** 95.5 ± 1.6683*** 115.8333 ± 3.0046*** 120 ± 1.3904*** t4:5 POSITIVE CONTROL 50.6 ± 0.6708 76.3333 ± 1.0540 80.3333 ± 0.4944## 86.6666 ± 0.8027## 90.5 ± 1.2041### t4:6 LC 400 mg/kg bw 50.6666 ± 0.7601 79.3333 ± 0.5577 86.8333 ± 0.9098# 91.3333 ± 0.9545## 96.166 ± 1.7779### t4:7 LC 200 mg/kg bw 52.1666 ± 0.9457 77.5 ± 1.3601 86.3333 ± 0.6666# 90.3333 ± 1.4981## 94.1 ± 0.4772### t4:8 LC 100 mg/kg bw 51.3333 ± 0.7601 80.1666 ± 0.4772 84.5 ± 0.7637# 92.3333 ± 0.9189## 97.6666 ± 0.7149### t4:9 FRC LC 200 mg/kg bw 53.333 ± 0.9888 76.5 ± 0.6191 83.3333 ± 0.8819# 88 ± 0.9309## 92.666 ± 0.7149###

*p <0.05,**p < 0.001, ***p < 0.0001 when compared with NORMALGROUP # p <0.05,## p <0.001,### p <0.0001whencomparedwithDISEASE CONTROL JrnlID 40200_ArtID 749_Proof# 1 - 31/01/2021

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t5:1 Table 5 Effect of Various Treatment Groups on Lipid profile

t5:2 GROUPS TC mg/dl TGmg/dl HDLmg/dl VLDLmg/dl LDLmg/dl

t5:3 NORMAL GROUP 112.3333±1.4529 81.8133±2.3004 27.5083 ± 0.9223 16.3583 ± 0.4602 68.3216 ± 1.1084 t5:4 DISEASE GROUP 149.8333±1.5793*** 151.5216±1.064*** 16.69 ± 0.9462*** 30.2983 ± 0.2131*** 102.7483 ± 11053*** t5:5 POSITIVE CONTROL 122.8333±1.2224### 94.678±1.3832### 26.9866 ± 0.7761### 18.9533 ± 0.2774### 77.2133 ± 1.3683### t5:6 LC 400 mg/kg bw 138.3333±2.4037## 97.4116±1.0187### 21.571 ± 0.8701## 19.4783 ± 0.2040*** 97.6883 ± 1.5985*** ### ### ### ### t5:7 LC 200 mg/kg bw 123.8333±1.0137 93.0366±1.048### 24.9283 ± 1.1206 18.5983 ± 0.2175 78.0633 ± 0.9150 t5:8 LC 100 mg/kg bw 131.8333±2.2718## 94.4366±1.1206### 22.8116 ± 0.7859## 18.805 ± 0.2449## 89.4616 ± 2.3383*** t5:9 FRC LC200 mg/kg bw 121.5±1.627### 92.2366±1.6120### 24.1866 ± 0.6934### 18.4433 ± 0.3223## 83.0566 ± 1.2917###

*p<0.05, **p<0.001, ***p<0.0001 when compared with NORMALGROUP # p<0.05, ## p<0.001, ### p<0.0001 when compared with DISEASE CONTROL

409 and spotty necrosis when compared to normal group mice to have anti- obesity properties. The selected dose of fraction- 428 410 having normal pathology (Fig. 1). On treatment with Luffa ation extract of saponins wasOF 200 mg/kg bw. 429 411 cylindrica extract and fractionation of saponins extract, there The experimental mice were induced into obesity by sub- 430 412 was recovery as compared to the progesterone-administered cutaneous administration of progesterone at 10 mg/kg. It can 431 413 group. However, treatment with the standard drug Orlistat 10 be inferred from previous research that progesterone, which is 432 414 mg/kg body weight showed cellular integrity comparable with a sex hormone widely used for contraceptive purposes HRT, 433 415 normal. There was an increase in the size of adipocytes in the induces hyperphagiaPRO by decreasing the serotonin level in the 434 416 progesterone-administered group compared to the normal brain. 435 417 control group on treatment with Luffa cylindrica extract and Progesterone,D a female reproductive hormone, is a neuro- 436 418 fractionation of saponins extract, with the adipocytes almost activeE steroid [19]. Women using depot medroxyprogesterone 437 419 nearing the same as normal (Fig. 2). have been noted to experience a substantial increase in weight 438 comprising entirely of increased fat mass and not lean mass 439 [31]. Furthermore previous studies have also suggested that 440 considerable weight gain is one of the side-effects of proges- 441 420 Discussion terone preparations intended for HRT or contraceptive use 442 because they tended to increase fat deposition. In fact, among 443 421 The present study was conducted on Swiss albino mice with all steroid hormones that promote synthesis and storage of fat, 444 422 progesterone-induced obesity to assess theORRECT potential activity of Progesterone is widely recognized as having the most obesity 445 423 Luffa cylindrica for anti-obesity effect. Luffa cylindrica ex- inducing effect. Therefore, weight gain and fat deposition 446 424 tract is known to contain phytoconstituentsC with LD50 value caused by progesterone induced hyperphagia is best suited 447 425 reported to be 2000 mg/kg bw. Hence, doses of 100, 200 and for an experimental model for research into hormone- 448 426 400 mg/kg bw were taken for current study. Traditionally, induced obesity. While it may be noted that the exact neuro- 449 427 Luffa cylindrica is believed toUN contain saponins that are known chemical mechanism of appetite regulation by progesterone is 450

t6:1 Table 6 Effect of Various Treatment Groups on SGOT and SGPT Table 7 Effect of Various Treatment Groups on Serotonin Levels t7:1

t6:2 GROUPS SGPT IU/L SGOT IU/L GROUPS SEROTONIN LEVELS t7:2

t6:3 NormalGroup 33.8633 ± 1.6667 44.0283±1.3251 NORMAL GROUP 0.82 ± 0.22 t7:3 t6:4 Disease group 78.98 ± 1.3968*** 85.815±1.2378*** DISEASE GROUP 0.38 ± 0.55### t7:4 t6:5 Positive control 47.8566 ± 2.0335### 55.7533±1.0844### POSITIVECONTROL 0.43 ± 0.11 t7:5 ## ## t6:6 LC 400 mg/kg bw 60.5483 ± 1.0921 68.135±9473### LC 400 mg/kg bw 0.6 ± 0.29 t7:6 ### ### t6:7 LC 200 mg/kg bw 48.92 ± 1.3011 63.065±1.372### LC 200 mg/kg bw 0.67 ± 0.45 t7:7 ### ## t6:8 LC100 mg/kg bw 52.0116 ± 1.6249 66.42±1.439## LC 100 mgkgl bw 0.57 ± 0.36 t7:8 ### ### t6:9 FRC LC200 mg/kg bw 46.6133 ± 1.5857 58.121±1.3176### FRC LC200 mg/kg bw 0.78 ± 0.22 t7:9

*p < 0.05, **p < 0.001, ***p < 0.0001 when compared with *p <0.05,**p < 0.001, ***p < 0.0001 when compared with NORMALGROUP # p < 0.05, ## p < 0.001, ### p < 0.0001 when com- NORMALGROUP # p < 0.05, ## p < 0.001, ### p < 0.0001 when com- pared with DISEASE CONTROL pared with DISEASE control JrnlID 40200_ArtID 749_Proof# 1 - 31/01/2021

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t8:1 Table 8 Effect of Various t8:2 Treatment Groups on hepatic GROUPS GSH CAT SOD enzymes t8:3 NORMAL GROUP 31.8 ± 1.3155 24.2683 ± 2.009 74.55 ± 1.2542 t8:4 DISEASE GROUP 17.1733 ± 1.2205*** 11.8766 ± 1.0899 ** 25.0783 ± 1.1360 ** t8:5 POSITIVE CONTROL 28.1336 ± 2.3758### 22.73 ± 1.3456### 70.72833 ± 1.318### t8:6 LC 400 mg/kg bw 24.6216 ± 1.1626## 20.4166 ± 1.0184## 63.606 ± 1.0019### t8:7 LC 200 mg/kg bw 28.6533 ± 2.0346### 22.67 ± 1.2986### 70.43 ± 1.0545### t8:8 LC 100 mg/kg bw 25.8888 ± 1.5239## 21.9366 ± 1.4633## 65.6266 ± 1.1556### t8:9 FRC LC2 00 mg/kg bw 30.0488 ± 0.8086### 23.144 ± 1.2231### 71.125 ± 1.3100###

*p <0.05,**p < 0.001, ***p < 0.0001 when compared with NORMALGROUP # p <0.05,## p <0.001,### p < 0.0001 when compared with DISEASE CONTROl

451 yet to be elucidated, the existing evidence suggests that it is administration of progesterone results in a central action on 476 452 the anti-estrogenic nature of progesterone that lies at the heart the receptors of progesterone located in the limbic system in 477 453 of increased food intake [19]. the hypothalamus bypassingOF blood- brain barriers. Regulation 478 454 A gradual increase in body weight has been confirmed of mood and emotions is influenced by the limbic system. 479 455 in the present study. Earlier research indicated that indi- In the present study a significant drop in exploratory be- 480 456 viduals administered with progesterone as part of treat- havior which include ambulation, rearing and grooming was 481 457 ment faced with increased body weight and in the total noted in the disease control group when compared to normal 482 458 proportion of body fat [32]. Luffa cylindrica extract at group. TreatmentPRO with Luffa cylindrica extract reversed the 483 459 dose 200 mg/kg bw when taken with progesterone result- effects on the exploratory behavior indicating an antidepres- 484 460 ed in a gradual decrease in body weight, BMI and food sant activityD of the plant extract. 485 461 consumption from the14th day, further fractionation ex- ESerotonin signaling in the hypothalamus is responsible for 486 462 tract of saponins demonstrated a significant attenuation the control/homeostatic regulation of food intake and the bal- 487 463 of body weight, BMI and food consumption. The previ- ance of resulting energy and is considered a critical brain 488 464 ous studies suggests the anti-obesity effect of medicinal mechanism.These serotonergic neurons are recognized as be- 489 465 plant may be due to its action on the hypothalamus or not ing as expressers of the progestin receptors. This leads us to 490 466 promote fat synthesis and storage. Also there was attenu- believe that the regulation of food intake and body weight 491 467 ation in the water intake on administration of both Luffa could be a result of a possible relationship between the sero- 492 468 cylindrical and fractionation extract of saponins indicating tonin receptor systems and the neurosteroid in the brain. There 493 469 the role of the extract either on the thirstORRECT centre or the is also available data implicating the impairment in the hor- 494 470 consequence of less food consumption. mone levels in the ovarian hormone predisposing eating dis- 495 471 Progesterone is also associated withC reduced anxiety. The orders in women as a consequence of inducing changes in the 496 472 hypothesis that progesterone acts directly on plasma mem- serotonin levels/serotonergic receptor function19. The present 497

473 brane receptors that are independent form of GABAA recep- study notes a decrease in the serotonin levels in the disease 498 474 tors is supported by the factUN that behavioral changes can be control group of the experimental models indicating the pos- 499 475 noted as early as 30 minutes after its administration. Peripheral sibility of hyperphagia. 500

t9:1 Table 9 Effect of Various t9:2 Treatment Groups on Blood GROUPS BLOOD PRESSURE BLOOD PRESSURE Pressure BEFORE TREATMENT AFTER TREATMENT

t9:3 NORMAL GROUP 110.1666 ± 1.3519 121.5 ± 1.2315 t9:4 DISEASE GROUP 110.1666 ± 1.5365 135.5 ± 1.2845 t9:5 POSITIVE CONTROL 110.3333 ± 1.4063 132.3333 ± 1.5202 t9:6 LC 400 mg/kg bw 107.6666 ± 1.3824 130.5 ± 1.1180 t9:7 LC 200 mg/kg bw 111.5 ± 1.1180 125 ± 1.2382 t9:8 LC 100 mg/kg bw 108.1666 ± 2.1819 128.5 ± 1.1761 t9:9 FRC LC 200 mg/kg bw 105.5 ± 0.6191 127.5 ± 1.0878

*p <0.05,**p < 0.001, ***p <0.0001whencomparedwithNORMALGROUP# p <0.05, ## p < 0.001, ### p < 0.0001 when compared with DISEASCONTROL JrnlID 40200_ArtID 749_Proof# 1 - 31/01/2021

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OF

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Fig. 1 Effect of Various Treatments on Histopathology of Liver at 40X. showing mild spotty necrosis and portal triditis. (d), LC 200 mg/kg bw: Normal group: Section of liver showing the normal intact architecture of Reduction in lipid droplets and mild piecemeal necrosis. (e), LC hepatocytes (a). Disease control Section of liver showing venous 100 mg/kg bw: Shows mild ballooning degeneration, apoptosis, and congestion, spotty necrosis, Kupffer cell hyperplasiaORRECT and inflammation hepatocellular dysplasia. (f), FRC LC 200 mg/kg bw: Minimal cellular (b), Positive control: Section of liver showing mild inflammation and death and sinusoidal congestion (g) Kupffer cell hyperplasia (c), LC 400 mg/kgC bw: Section of liver UN 501 Administration of Luffa cylindrica extract and fractionation exists experimental evidence indicating an increased re- 517 502 extract of saponins at a dose of 200 mg/kg bw demonstrated a lease of insulin which means increased levels of blood 518 503 rise in serotonin levels suggesting that Luffa cylindrica may glucose on the administration of progesterone. 519 504 affect the 5-HT2C receptors and serotonin levels. In the present study, a significant rise in the serum 520 505 Increased secretion of insulin may be related to the levels of TG, TC, LDL, VLDL and blood glucose and a 521 506 anabolic effects of progesterone. Excess insulin secretion decrease in HDL levels was noted on the administration 522 507 in such circumstances could be predicted to support great- of progesterone as compared to normal mice confirming 523 508 er lipogenesis in the liver as well as adipose tissue and the occurrence of hyperlipidemia and hyperglycemia. 524 509 secondly, cause a rise in the fatty acids available for the Serum TG, TC, LDL, VLDL were significantly atten- 525 510 biosynthesis of triglycerides by supporting the production uatedwithanincreaseinHDLcholesterolaswellas 526 511 of lipoprotein lipase and thirdly, impair lipolysis. A direct lowering of glucose levels on Treatment with Luffa 527 512 effect of estrogen and progesterone on the beta cellules of cylindrica and fractionation of saponins 3extract at dose 528 513 the islets of Langerhans is postulated due to the presence of 200 mg/kg bw in comparison with the disease con- 529 514 of receptors in these cellules. Sex hormones; especially, trol group of experimental models. This demonstrates 530 515 progesterone (and synthetic progestins used in contracep- the hypolipidaemic and hypoglycaemic effect of plant 531 516 tion) appear to have a crucial role to play here. There extracts on the mitigation of obesity. 532 JrnlID 40200_ArtID 749_Proof# 1 - 31/01/2021

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Fig. 2 Effect of Various Treatments on Adipocyte Size at 40X. Normal tissue showing largest adipocyte size 102 μM(d); LC 200 mg/kg bw: group: Section ofiadipose tissue showing largest adipocyte size 62 μM Section of adipose tissue showing largest adipocyte size 80 μM(e); LC (a); Disease control Section of adipose tissue showing largest adipocyte 100 mg/kg bw: Section of adipose tissue showing largest adipocyte size size 114 μM(b); Positive control: Section of adipose tissue showing 95 μM(f); FRC LC 200 mg/kg bw: Section ofiadipose issue showing largest adipocyte size 82 μM(c); LC 400 mg/kg bw: Section of adipose largest adipocyte size 73 μM(g)

533 Progesterone administration is knownORRECT to cause an increase antioxidant content present in extracts of vegetables of the 554 534 in SGPT and SGOT levels which isC an indication of hepatic gourd family can be ascribed to their being particularly phenol 555 535 injury or abnormality. In the present study, a significant in- and flavonoid-rich in nature. 556 536 crease in SGPT and SGOT levels and reduction in antioxidant There has been previous research that has reported marked 557 537 biomarkers i.e., SOD, CAT,UN GSH was noted in experimental hypertrophy of the hepatic smooth surface endoplasmic retic- 558 538 mice that were administered progesterone. This could be a ulum on repeated administration of progesterone, raising the 559 539 result of different types of non-oxidized, free radicals being possibility of steroids being physiologic inducers as well as a 560 540 generated in the human body all the time for carrying out natural substrate of the enzymes of the smooth reticulum [33]. 561 541 particular metabolic functions and being eliminated by an ac- The current study noted a significant increase in liver weight 562 542 tive mechanism of antioxidants at work in the same. If the with venous congestion, sinusoidal congestion and Kupffer 563 543 production of these free radicals is more than the eliminating cell hyperplasia on the administration of progesterone as com- 564 544 capacity of the antioxidant network, it causes oxidative dam- pared to normal group mice, indicating the induction of 565 545 age to biomolecules and tissues, eventually causing degener- hypertrophy. 566 546 ative and autoimmune diseases. Administration of Luffa cylindrica extract significantly de- 567 547 Treatment with Luffa cylindrica extracts and fractionation creased the liver congession as compared to normal mice dem- 568 548 extract of saponins during the current study, in dosages of 200 onstrating that the extracts has a hetaptoprotective property. 569 549 mg/ kg bw, significantly increased the amounts of antioxi- One of the important consideration to be fulfilled for the 570 550 dants, as well as demonstrated a significant rise in the GSH, present study is to network pharmacology of the Luffa 571 551 SOD, CAT elements present in the blood. Luffa cylindrica is cylindrica as explained by Khanal et al [34–36], Patil et al 572 552 known to be rich in a variety of phytochemicals to varying [37] and Duyu et al [38] which could help in identifying the 573 553 extents, for eg, carotenoids, flavonoids, and phenols. The rich probable interaction with the targets related to obesity. 574 JrnlID 40200_ArtID 749_Proof# 1 - 31/01/2021

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575 Secondly, it was reported to the multiple biological profiles of mechanism is not elucidated. The extracts have also resulted 625 576 a single hits of secondary metabolites as explained previously in significant alterations to the liver as was obvious from his- 626 577 [39], which could be performed for Luffa cylindrical also. topathology. Due to its prominent anti-obesity activity, the 627 578 Progestins have an enlarging effect on the size of the adi- extract can be used for adjuvant therapy in the treatment of 628 579 pocyte cells but do not affect the numbers of these cells. hyperphagic effect of progesterone-induced obesity, however, 629 580 Stimulated production of the enzyme lipoprotein lipase which this needs further clinical evaluation. 630 581 is responsible for circulating plasma triglyceride hydrolysis 582 and fat cell absorption by FFAs produced during this process, 583 can explain the enhancement of depot triglyceride storage. Abbreviations BMI, Body Mass Index; BW, Body Weight; TG, 631 632 584 The histological findings from the current study reveal a Triglycerides; TC, Total Cholesterol; HDL, High Density Lipoprotein; LDL, Low Density-Lipoprotein; VLDL, Very Low Density Lipoprotein; 633 585 significant increase in the number and size of adipose cells SGPT, Serum Glutamic Pyruvic Transaminase; SGOT, Serum Glutamic 634 586 (largest size 114.65 μm and 15 adipocytes/40000 square mi- Oxaloacetic Transminase; LC extract, Luffa cylindrical extract; Fraction 635 587 cron) caused by the administration of progesterone as well as of LC, Luffa cylindrical extract fraction group 636 637 588 congestion which is per previous studies. Treatment with Acknowledgements The authors are thankful to Prof. Dr. S. S. Jalalpure 638 589 Luffa cylindrica extracts significantly decreased the number Principal, KLE college of Pharmacy, Belagavi, Karnataka, India for pro- 639 590 and size of adipose cells and congestion (53.83μm) compared viding necessary facilities, I thank KAHER for cosidering for research 640 591 to the cell size of progesterone-administered mice.This indi- funding the project. OF 641 592 cates the extracts have a beneficial effect on obesity. 642 ’ 643 593 After a thorough study of in vivo results, it can be hypoth- Authors contributions All the Authors are designed equally involved in the study and the experiments and Reviewed the written drafted the man- 644 594 esized that extracts of Luffa cylindrica and fractionation ex- uscript. Contributed in conduct of the experiments, performed data anal- 645 595 tract of saponins have an anti-obesity effect on mice. The ysis. The author(s)PRO read and approved the final manuscript. 646Q5 596 results of the present study demonstrate that Luffa cylindrica 597 extract along with fractionation of saponins at a dose of 200 DeclarationsD 647 598 mg/kg body weight results in a significant anti-obesity effect E 599 on progesterone-induced obesity in female Swiss albino mice. Conflict of interest The authors declare no conflict of interest. 648 600 The main limitation of the present study is that there is no 601 estimation of metabolites which could have influence the out- 602 come of the study and the present study mainly focused the References 649 603 anti-obese activity of the L. cylindrica for the first time. 650 604 Further the proper evaluation is to be made to proper molec- 1. Grundy SM. Obesity, metabolic syndrome, and cardiovascular dis- ease. J Clin Endocrinol Metab. 2004;89(6):2595–600. 651 605 ular mechanism of the extract via the bioactivity guided eval- 2. Lavie CJ, McAuley PA, Church TS, Milani RV, Blair SN. Obesity 652 606 uation and identifying the relevant regulated biomarkers. and cardiovascular diseases: implications regarding fitness, fatness, 653 ORRECT and severity in the obesity paradox. J Am Coll Cardiol. 654 2014;63(14):1345–54. 655 C 656 607 Conclusion 3. Piché M-E, Poirier P, Lemieux I, Després J-P. Overview of Epidemiology and Contribution of Obesity and Body Fat 657 Distribution to Cardiovascular Disease: An Update. Prog 658 608 Progesterone injection (10mg/kg)UN demonstrated a highly sig- Cardiovasc Dis. 2018;61:103–13. 659 609 4. Ng M, Fleming T, Robinson M, Thomson B, Graetz N, Margono C, 660 nificant increase in weight. The study exhibits potential mol- 661 610 et al. Global, regional, and national prevalence of overweight and ecule of Luffa cylindrica to interact with the majority of pro- obesity in children and adults during 1980 2013: a systematic anal- 662 611 teins that are implicated in the pathogenesis obesity. Oral ad- ysis for the global burden of disease study 2013. Lancet. 2014;384: 663 612 ministration of hydro-alcoholic extract of Luffa cylindrica for 766–81. 664 665 613 28 days lowered the levels of circulating lipids 5. Keys A, Fidanza F, Karvonen MJ, Kimura N, Taylor HL. Indices of – 666 614 relative weight and obesity. Int J Epidemiol. 2014;43(3):655 65. inthebloodalongsidereducingthediameteroftheadipocytecells- 6. Poirier P, Alpert MA, Fleisher LA, Thompson PD, Sugerman HJ, 667 615 causingfemaleswissalbino mice to lose body weight, which Burke LE, et al. Cardiovascular evaluation and management of 668 616 bears a striking similarity to obesity patterns in human fe- severely obese patients undergoing surgery: a science advisory 669 617 males. Along with this, the extract revealed the influence it from the American Heart Association. Circulation. 2009;120(1): 670 86–95. 671 618 exerts on increasing serotonin levels in the brain and also on 7. Poirier P, Giles TD, Bray GA, Hong Y, Stern JS, Pi-Sunyer FX, 672 619 increasing the exploratory behavior which was decreased in et al. Obesity and cardiovascular disease: pathophysiology, evalu- 673 620 progesterone-administered mice. The extract also showed re- ation, and effect of weight loss: an update of the 1997 American 674 621 duced glucose levels and improved antioxidant content. The Heart Association scientific statement on obesity and heart disease 675 676 622 anti-obesity potential of the Luffa cylindrica plant could be a from the obesity Committee of the Council on nutrition, physical activity, and metabolism. Circulation. 2006;113(6):898–918. 677 623 consequence of its phytochemical content which includes fla- 8. Wormser D, Kaptoge S, Di Angelantonio E, Wood AM, Pennells 678 624 vonoids, saponins, alkaloids, tannins, steroids, but the exact L, Thompson A, et al. Separate and combined associations of body- 679 JrnlID 40200_ArtID 749_Proof# 1 - 31/01/2021

J Diabetes Metab Disord

680 mass index and abdominal adiposity with cardiovascular disease: 26. Nderitu KW, Mwenda NS, Macharia NJ, Barasa SS, Ngugi MP. 737 681 Emerging Risk Factors collaborative analysis of 58 prospective Antiobesity activities of Methanolic extracts of Amaranthus dubius, 738 682 studies. Lancet. 2011;377(9771):1085–95. Cucurbita pepo, and Vigna unguiculata in progesterone-induced 739 683 9. Bastien M, Poirier P, Lemieux I, Després JP. Overview of epide- obese mice. Evid Based Complement Alternat Med. 2017;2017: 740 684 miology and contribution of obesity to cardiovascular disease. Prog 4317321. 741 685 Cardiovasc Dis. 2014;56(4):369–81. 27. Gargiulo S, Gramanzini M, Megna R, Greco A, Albanese S, 742 686 10. Asarian L, Geary N. Modulation of appetite by gonadal steroid Manfredi C, et al. EvaluationofGrowthPatternsandBody 743 687 hormones. Philos Trans R Soc Lond Ser B Biol Sci. Composition in C57Bl/6J Mice Using Dual Energy X-Ray 744 688 2006;361(1471):1251–63. Absorptiometry. BioMed Res Int. 2014;2014(253067):11. https:// 745 689 11. Carlson MJ, Thiel KW, Yang S, Leslie KK. Catch It Before It Kills: doi.org/10.1155/2014/253067. 746 690 Progesterone, Obesity, and the Prevention of Endometrial Cancer. 28. Lipid estimation, Modified Roechlau’s method, manufactured by 747 691 Discov Med. 2012;14(76):215–22 PMCID: PMC3964851. Ebra mannheim gmbh kit, marketed by transasia Bio-Medicals Ltd. 748 692 12. Amatayakul K, Sivasomboon B, Thanangkul O. A study of the 29. Enzyme estimation by, Erba Mannheim gmbh kit, marketed by 749 693 mechanism of weight gain in medroxyprogesterone acetate users. transasia Bio-Medicals Ltd. 750Q6 694 Contraception. 1980;22(6):605–22. 751 695 30. Schlumpf M, Lichtensteiger W, Langemann H, Waser PG, Hefti F. 13. Wallace JM, Shively CA, Clarkson TB. Effects of hormone re- A fluorometric micromethod for the simultaneous determination of 752 696 placement therapy and social stress on body fat distribution surgi- 753 697 – serotonin, noradrenaline and dopamine in milligram amounts of cally postmenopausal monkeys. Int J Obes. 1999;23:518 27. brain tissue. Biochem Pharmacol. 1974;23(17):2437–46. 754 698 14. Bäckström T. Symptoms related to the menopause and sex steroid 31. Clark MK, Dillon JS, Sowers M, Nichols S. Weight, fat mass, and 755 699 treatments. CIBA Found Symp. 1995;191:171–80 discussion 180- central distribution of fat increase when women use depot 756 700 6. OF medroxyprogesterone acetate for contraception. Int J Obesity. 757 701 15. Crammer JL. Premenstrual depression, cortisol and oestradiol treat- 2005;29:1252–8. 758 702 ment. Psychol Med. 1986;16(2):451–5. 703 32. Shirling D, Ashby JP, Baird JD. Effect of progesterone on lipid 759 16. Bethea CL, Gundlah C, Mirkes SJ. Ovarian steroid action in the – 760 704 serotonin neural system of macaques. Novartis Found Symp. metabolism in the intact rat. J Endocrinol. 1981;90(2):285 94. 761 705 2000;230:112–30. 33. Emans JB, Jones AL. Hypertrophy of liver cell smooth surfaced PRO 762 706 17. Kaur G, Kulkarni SK. Evidence for serotonergic modulation of reticulum following progesterone administration. J Histochem – 763 707 progesterone-induced hyperphagia, depression and algesia in fe- Cytochem. 1968;16(9):561 71. https://doi.org/10.1177/16.9.561. 708 male mice. Brain Res. 2002;943(2):206–15. 34. KhanalD P, Patil BM. Integration of network and experimental phar- 764 709 18. WHO. Obesity: preventing and managing the global epidemic. Emacology to decipher the antidiabetic action of Duranta repens L. J 765 710 WHO Technical Report Series number 894. Geneva: WHO; 2000. Integr Med. 2020;S2095–4964(20):30109–6. https://doi.org/10. 766 711 19. Lee YS, Cha BY, Saito K, Choi SS, Wang XX, Choi BK, et al. 1016/j.joim.2020.10.003 Epub ahead of print. 767 712 Effects of a Citrus depressa Hayata (shiikuwasa) extract on obesity 35. Khanal P, Patil BM, Chand J, Naaz Y. Anthraquinone Derivatives 768 713 in high-fat diet- induced obese mice. Phytomedicine. 2011;18(8–9): as an Immune Booster and their Therapeutic Option Against 769 714 648–54. COVID-19. Nat Prod Bioprospect. 2020;10(5):325–35. https:// 770 715 20. Murase T, Misawa K, Minegishi Y, Aoki M, Ominami H, Suzuki doi.org/10.1007/s13659-020-00260-2 Epub 2020 Aug 8. PMID: 771 716 Y, et al. Coffee polyphenols suppress diet-induced body fat accu- 32772313; PMCID: PMC7414902. 772 717 mulation by downregulating SREBP- 1c and related molecules in 36. Khanal P, Duyu T, Patil BM, Dey YN, Pasha I, Wanjari M, et al. 773 718 C57BL/6J mice. Am J Physiol Endocrinol Metab. 2011;300(1): Network pharmacology of AYUSH recommended immune- 774 719 E122–33. boosting medicinal plants against COVID-19. J Ayurveda Integr 775 720 21. Gupta N, Hallman DM, Dumuid D, VijORRECT A, Rasmussen CL, Med. 2020. https://doi.org/10.1016/j.jaim.2020.11.004 Epub 776 721 Jørgensen MB, et al. Movement behavior profiles and obesity: a ahead of print. PMID: 33250601; PMCID: PMC7687402. 777 722 latent profile analysis of 24-h time-useC composition among Danish 37. Patil R, Chikhale R, Khanal P, Gurav N, Ayyanar M, Sinha S, et al. 778 723 workers. Int J Obes. 2020;44:409–17. https://doi.org/10.1038/ Computational and Network Pharmacology Analysis of 779 724 s41366-019-0419-8. Bioflavonoids as Possible Natural Antiviral Compounds in 780 725 22. AZEEZ MA, BELLO OS,UN Omobola A. Traditional and medicinal Covid-19. Inform Med Unlocked. 2020;22:100504. https://doi. 781 726 uses of Luffa cylindrica :a Review. J Med Plants Studies. 2013;1(5): org/10.1016/j.imu.2020.100504. 782 727 102–11. 38. Duyu T, Khanal P, Khatib NA, Patil BM. Mimosa pudica modu- 783 728 23. Partap S, Kumar A, Sharma NK, Jha KK. Luffa Cylindrica: An lates Neuroactive ligand-receptor interaction in Parkinson’s disease. 784 729 important medicinal plant. J Nat Prod Plant Resour. 2012;2(1): Indian J Pharm Educ Res. 2020;54(3):732–9. 785 730 – 127 34. 39. Khanal P, Patil BM. In vitro and in silico anti-oxidant, cytotoxicity 786 731 24. Khare CP. Indian medicinal plants: an illustrated dictionary. and biological activities of Ficus benghalensis and Duranta repens. 787 732 Springer Science & Business Media; 2008. Chinese Herbal Med. 2020;12(4):406–13. 788 733 25. Khanal P, Patil BM. Gene ontology enrichment analysis of α- 734 amylase inhibitors from Duranta repens in diabetes mellitus. J 735 Publisher’snote Springer Nature remains neutral with regard to jurisdic- 789 Diabetes Metab Disord. 2020. https://doi.org/10.1007/s40200- 790 736 020-00554-9. tional claims in published maps and institutional affiliations. 791 792 AUTHOR QUERIES

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