Evaluation of Rhizome Extract of Zingiber officinale for Anti-Obesity Property in Wistar Rats

Obesity is a chronic complex disease defined as an abnormal or excessive fat accumulation that presents a risk to health. The main cause of weight gain and obesity is the imbalance between the number of calories an individual takes in and the calories the individual burns ^{1, 2}. Over the last century, obesity has emerged as a leading global health concern through recent environmental and societal changes favouring a positive energy balance and weight gain. The main factors are the consumption of high-calorie or high-fat foods, insufficient physical activity, and a shift towards a well-developed sedentary lifestyle. Consequently, the prevalence of obesity nearly doubled worldwide since 1980. In 2014, more than 39% of the adults, who were 18 years or older, were overweight with 13?ing obese. In addition, at least 41 million children under the age of 5 were overweight or obese ². In 2022, 1 in 8 people in the world were living with obesity. Worldwide, adult obesity has more than doubled since 1990, and adolescent obesity has quadrupled. In 2022, 43% of adults (18 years and older) were overweight. Of these, 16% were living with obesity. In 2022, 37 million children under the age of 5 were overweight. Over 390 million children and adolescents aged 5 – 19 years were overweight, including 160 million who were living with obesity ². Severe obesity i.e., Body Mass Index (BMI) > 35 kg/m² is nowadays a rapidly growing segment of the global epidemic, which is especially marked by its negative effect on health as an increase in BMI implies an elevated mortality risk, like low BMI does. Nonetheless, overweight and obesity are killing more people today than underweight ³. Obesity is known to be the main risk factor for several non-communicable diseases like cardiovascular disease, hypertension, coronary heart disease, type 2 diabetes, nonalcoholic fatty liver disease, polycystic ovary disease, obstructive sleep apnoea, gallbladder disease, osteoarthritis, pain, gastroesophageal reflux disease, incontinence, lymphoedema, plantar fasciitis or certain types of cancers (colon, kidney, oesophageal, postmenopausal breast and endometrial). Obesity also causes diverse psychological problems or various physical disabilities. It also influences the quality of living, such as sleeping or moving. Other potential complications include mood, anxiety and eating disorders. Obesity can reduce healthy life years and can reduce life expectancy by 6 – 14 years ^{2, 4,5}. Treatment for obesity aims at helping obese individuals reach and stay at a healthy weight, limit their risks of developing other serious health issues, and improve their overall quality of life. Management of obesity includes non-surgical methods such as dieting, exercise or physical therapy and pharmacological therapy. The safety and long-term success of several anti-obesity surgeries have been demonstrated ⁶. Several researchers have demonstrated the potential of natural products and their bioactive compounds for treating obesity. This may be a good alternative for developing future and safe anti-obesity drugs. A vast number of natural products, including crude extracts and isolated bioactive compounds from plants can induce body weight reduction and prevent diet-induced obesity. They have, therefore, been widely used in the treatment of obesity ⁷.  Zingiber officinale (commonly called Ginger) belongs to the family Zingiberaceae ⁸. It is a tropical plant which grows well in hot and humid climates. The plant is cultivated in China, Nepal, the US, India, Bangladesh, Taiwan, Jamaica, Nigeria and Indonesia ⁹. Zingiber officinale is an erect perennial plant growing from one to three feet in height. The stem sticks up about 12 inches above the ground and is surrounded by the sheathing bases of the two-ranked leaves. It produces clusters of white and pink flower buds that bloom into yellow flowers. Zingiber officinale grows horizontally, laterally flattened with branching pieces, a configuration known as rhizome. The whole rhizome has a firm, striated texture. It is 5 to 15cm long, 1.5 to 6 cm wide, 2 cm thick and depending on the variety can be yellow, white, or red.  Zingiber officinale are used as part of Traditional Medicine Systems and there is a large body of anecdotal evidence supporting their use and efficacy. It is used to treat headaches, nausea, colds, arthritis, rheumatism, muscular discomfort, and inflammation. It is used for reducing pain, rheumatoid arthritis, and it has antioxidant effects ^{10, 11,12}. It is used as therapy against several cardiovascular diseases (such as hypertension, atherosclerosis, cholesterol), gastrointestinal disorders, respiratory disorders, migraine, depression, gastric ulcers and is anti-emetic ^{13, 14, 15, 16, 17}. In Nigeria, Z. officinale is believed to have an anti-obesity effect.   This study was aimed at evaluating the aqueous-methanol extract of Zingiber officinale rhizome for anti-obesity effect in Wistar rats with the view of authenticating the claim and possibly developing it as an anti-obesity drug that is standardized and readily made available for obese individuals.
MATERIALS AND METHODS

Plant Collection and Identification

Zingiber officinale (Ginger) rhizomes were collected from Central Market, kubwa, Bwari Area Council of Abuja, Nigeria. Kubwa has a latitude of 9.159634, longitude of  7.338608, Longitude DMS of  20’18.99” E Latitude DMS: 9’34.68” N. The samples were collected in the month of March. Identification and authentication were done by Mr Lateef Adamu, a Plant Taxonomist with the Department of Medicinal Plants Research and Traditional Medicine (MPR & TM), National Institute for Pharmaceutical Research and Development (NIPRD), Idu, Abuja, Nigeria.  The specimen was identified to be of the family Zingiberaceae with the Scientific name: Zingiber officinale. Voucher specimen no: NIPRD/H/7300 was assigned to the specimen.

Preparation of Crude Extract

Zingiber officinale rhizome was dried under a shade in a well-ventilated environment for 21 days. It was then pulverized using mortar and pestle, it was further ground into powder using a milling machine. 200 g of the pulverized rhizome was macerated in 80% methanol for 72 h. The mixture was filtered using filter paper and the filtrate was concentrated using a water bath at 40 ?. The extract was then kept in a refrigerator at 4?C until when needed for the study.

Determination of the Yield of the Extract

The resultant yield was determined after the extracted concentrate was weighed. The percentage yield was calculated using the given formula;

Percentage yield of extract (%) = weight of extract / weight of pulverized Z.O    X 100

Where Z.O Zingiber officinale.

Chemicals and Drugs

The drugs and chemicals used include Xenicals (120 g, each capsule contains 2s, 3s-3-hexyl-oxo-2=oxetomyl (methyl dodecyl esters) manufactured by R.S No. 63/3, and 4. Thirvander Koil, Pondicherry-605, 102102 India), Normal Saline, Methanol (Sigma- Aldrich, Darmstadt, Germany) and Cyproheptadine (Asset Pharmacy, Lagos, Nigeria).

Experimental Animals        

Wistar rats of both sexes were obtained from the Animal Facility Centre of the National Institute for Pharmaceutical Research and Development (NIPRD), Idu, Abuja, Nigeria. The rats were acclimatized for one week at the Animal Holding Section of the University of Abuja Veterinary Teaching Hospital. They were fed ad libitum daily with growers’ mash and allowed access to clean drinking water. They were maintained under 12 h day and night illumination cycle.

Acute Toxicity Study (LD₅₀)

The Acute toxicity study was done according to the modified method of Lorke ¹⁸. The method estimates the dose of the extract that will result in the death of 50 % of the treated animal population. The study was done in a biphasic manner. In the first phase, the following graded dosages of the plant extract were administered to three groups of rats (n = 3): 10, 100, and 1000 mg/kg orally respectively. In the second phase: graded doses of 2000, 3000 and 5000 mg/kg of the extract were administered orally to another three groups of rats (n=3) following observations of rats in the first phase. After administration, the rats were observed for 72 h for signs of toxicity such as nervousness, ataxia, excitement, writhing or death.

Anti-Obesity Study

Wistar rats of both sexes with mean weights between 173.2 g – 287.2 g were used for the study. Cyproheptadine (16 mg/kg) was administered intraperitoneally to all the rats to stimulate their appetite. The rats were then marked and separated into 5 groups (with 5 rats in each group) as follows:
Group A- Rats in this group were administered normal saline (10 mg/kg P.O) to serve as the 

negative control.

 Group B- Rats were administered aqueous-methanol extract of Zingiber officinale rhizome 

(100 mg/kg P.O).

Group C- Rats were administered an aqueous-methanol extract of Zingiber officinale rhizome

(200 mg/kg P.O).

Group D- Rats were administered an aqueous-methanol extract of Zingiber officinale rhizome

(400 mg/kg P.O).

Group E- Rats in this group were administered Xenicals (Orlistat^®; 12 mg/kg P.O) to serve as

the positive control group. The treatments lasted for 28 days during which the water intake, feed intake and body weight of all the rats were measured. 

Measurement of Water Intake

 A volume of 400 ml of water was measured with a measuring cylinder and poured into the drinkers for each group daily. The volume of water taken by the rats daily was calculated using the difference between the 400 ml of water given the previous day and the volume of water remaining in the current day of observation.

Measurement of Feed Intake

250 g of pelleted feed was weighed using a weighing balance and placed in the feeding troughs of each group of rats daily. The amount of feed consumed daily was calculated by finding the difference between the 250 g of the feed given the previous day and the feed remnant on the current day of observation.     

Measurement of Body Weight

 The body weights of the rats were taken on Day1 and then taken every week (weeks 1, 2, 3 and 4) using a weighing balance. The difference between the body weight of rats in each group was compared statistically with those of the negative control. The difference in the body weight of each group at the end of every week and its body weight on Day 1 was calculated and statistically analyzed.

The difference between the body weight of each group at the end of a prevailing (current) week and the previous week was also calculated and analyzed statistically.   The total weight gain for each group was determined using the difference in the body weight of each group on Day 28 and Day 1.

Statistical Analysis: All data were analyzed statistically by one-way analysis of variance (ANOVA) using the IBM SPSS version 23. Data were expressed as mean ± standard error of mean and P values ? 0.05 were considered statistically significant. Results were expressed as tables and a chat.

RESULTS

Extract Yield and Description

 The weight of the pulverized Zingiber officinale was 680 g, while the extract yield was 56 g.

The percentage yield was then calculated to be 8.2%. The extract obtained was sticky in texture and dark brown in colour.

Acute Toxicity Study

No mortality was recorded 72 h after the treatment of rats with the aqueous-methanol extract of Zingiber officinale rhizome at (10-5000 mg/kg P.O). However, rats treated with a 5000 mg/kg dose showed writhing reflex, which lasted for about 5 minutes. The oral (LD₅₀) of the extract was therefore estimated to be greater than 5000 mg/kg (Table 1).

Table 1: Determination of oral median lethal dose (LD₅₀) of aqueous-methanol extract of

Zingiber officinale rhizome in rats

n=3

Anti-Obesity Study

Effect of Aqueous-Methanol Extract of Zingiber officinale Rhizome on Water Intake of Rats Treated for 28 Days

The aqueous-methanol extract of Zingiber officinale rhizome (100, 200, and 400 mg/kg P.O) dose-dependently reduced water intake of rats within each week of the treatment period of 28 days (within week 1, week 2, week 3 and week 4). In week 1, the reduction within was significant (p < 0>®, 12 mg/kg P.O) within each of the weeks (Table 2).

Table 2: Effect of aqueous-methanol extract of Zingiber officinale rhizome (100, 200 and

400 mg/kg P.0) on water intake of rats treated for 28 days

Values are expressed as mean ± SEM; *P ? 0.05 = Significantly different from the control one-way ANOVA; Turkey post hoc.

Effect of Aqueous -Methanol Extract Zingiber officinale Rhizome on Feed Intake of Rats Treated for 28 Days

The aqueous-methanol extract of Zingiber officinale (100, 200, and 400 mg/kg P.O) caused a reduction in feed intake of rats within each of the weeks of the treatment period (i.e. within week 1, week 2, week 3 and week 4).  The feed reduction effect was dose-dependent and the reduction was significant (P < 0> at all the tested doses of 100 mg/kg P.O, 200 mg/kg P.O and 400 mg/kg P.O in weeks 2, 3 and 4. The reduction was also comparable to the reduction caused by Xenical (12 mg/kg P.O) which served as the positive control (Table 3).

Table 3: Effect of aqueous-methanol extract of Zingiber officinale rhizome (100, 200, and

400 mg/kg) on Feed Intake of rats Treated for 28 days

Values are expressed as mean ± SEM; *P ? 0.05 = significantly different from the control one-way ANOVA; Turkey post hoc.

Effect of Aqueous-Methanol Extract of Zingiber officinale Rhizome on the Body Weight of Rats Treated for 28 Days

The aqueous-methanol extract of Zingiber officinale (100, 200 and 400 mg/kg P.O) significantly (P < 0>

Table 4: Effect of aqueous-methanol extract of Zingiber officinale rhizome (100, 200 and

400 mg/kg P.0) on body weight of rats treated for 28 days

Values are expressed as mean ± SEM; *P ? 0.05 = significantly different from the control one-way ANOVA; Turkey post hoc.

Table 5: Effect of aqueous-methanol extract of Zingiber officinale rhizome (100, 200 and

400 mg/kg P.0) on body weight difference between current week and Day 1 of the same group of rats

Values are expressed as mean ± SEM; *P ? 0.05 = significantly different from the control one-way ANOVA; Turkey post hoc (D = Day).

Table 6: Effect of aqueous-methanol extract of Zingiber officinale rhizome (100, 200 and

400 mg/kg P.0) on body weight difference between prevailing/current week and previous week in the same groups

Values are expressed as mean ± SEM; *P ? 0.05 = significantly different from the control one-way ANOVA; Turkey post hoc (D = Day).

       
           
       
Figure 1: Total weight gain of rats treated with aqueous-methanol extract of Zingiber officinale rhizome (100, 200, and 400 mg/kg P.O) for 28 days (N/S= Normal Saline; Z.O=Zingiber officinale).