Effect of an herbal extract Number Ten (NT) on body weight in rats

The herbal decoction NT consists of a combination of 40% Rheum officinale Baill. (Dahuang), 13.3% Astragalus complanatus R. Br. (Shayuanjili), 13.3% Salvia miltiorrhiza Bge. (Danshen), 26–27% Curcuma longa L. (Jianghuang) and 6–7% Zingiber officinale Rosc. (Shengjiang). The rhubarb root and stem were placed in a stainless steel pot with water 6–8 times the herb's weight and a mixture of the other four herbs were prepared in a similar manner. The herbs were allowed to soak for eight hours. The water in the pot with the four-herb mixture was boiled until the volume was reduced by half. A cold rhubarb decoction was then added to the four-herb mixture and heated to just below boiling point for 20 minutes before cooling. After filtering the large particulates from the decoction, the remaining liquid was freeze-dried to a powder form, producing 0.5 g of solids from 10 ml of liquid.

Sixty female Wistar rats (with an average weight of 220 g at the beginning of the study) were housed individually in hanging wire mesh cages. Room temperature was maintained at 22–24°C and light cycle was 12 hours with lights out at 18:00. The animals were accustomed to handling and daily gastric intubation over a one week period.

Rats were divided into five experimental groups. All were placed onto a high fat diet with 29% protein, 24% carbohydrate and 30% fat by weight for 56 or 70 days (energy density of diet = 4.78 kcal/g). Fresh food was placed in the cages immediately before lights out. The five groups of rats were tube-fed between 16:00 and 18:00 daily before lights out with water (Control, 15 animals), 1.5 g of freeze-dried NT powder (NT-H, 15 animals), 0.75 g of freeze-dried NT powder (NT-L, 10 animals), water while pair fed to NT-H (Pr-fed, 15 animals) and 2 mg/kg of d-fenfluramine (d-FF, 10 animals) respectively. The experimental protocol was approved by the Institutional Animal Care and Use Committee and conformed to all the federal guidelines.

Food intake was recorded daily throughout the experiment; body weight was recorded daily for the first 23 days and twice a week from day 23 to day 56. Except for 5 animals in the control and the 1.5 g NT groups, all rats were sacrificed by guillotine to collect trunk blood on day 56. The remaining ten animals were sacrificed on day 70. The parametrial and retroperitoneal fat pads were carefully dissected free of surrounding tissues by a single investigator to ensure consistency of dissection. Other tissues were individually dissected and weighed in a similar manner.

Trunk blood was collected from fed rats between 10:00 and 14:00 and serum was prepared. Serum cholesterol, triglycerides, glucose, sodium, potassium, chloride, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, creatine phosphokinase, gamma glutamyl transpepdidase, amylase were assayed on a Beckman Coulter Synchron CX7 chemistry analyzer using standard methods. Rat insulin, leptin and corticosterone were assayed by commercial radioimmunoassays (Linco Corp, San Diego, California, USA). Platelets, white blood cell count, mean cell volume, hematocrit and hemoglobin were also assayed on a Beckman Coulter HMX.

During the 56 days of the experiment, all rats were observed daily by an animal technician, and weekly by a veterinarian who was blind to the individual treatments of each rat. Notations were made regarding their physical activity/lethargy, fur condition, skin condition, eye condition, stools and 'others'.

Body composition of the eviscerated carcasses was measured. The frozen carcasses were allowed to warm to room temperature and then dried in a 60°C oven to constant weight. The dried carcasses were homogenized repeatedly to produce a homogeneous sample. A sample of this homogenate was used to assay protein (Kjeldahl analysis), fat (Soxhlet extraction) and ash (combustion) for calculation of body composition.

Data were analyzed using the Statistical Package for Social Sciences (SPSS) version 12. Weight, food consumption, body composition, organ weights and clinical blood analytes were analyzed using one-way analysis of variance (ANOVA) followed by a Scheffe Comparison of Means analysis for unbalanced groups. After sample sizes were balanced, an additional Bonferroni and Tukey Comparison of Means analysis was performed. Feed efficiency was calculated from the amount of weight gained in grams per gram of food consumed. Correlation was estimated by Pearson's coefficients. The results with P values less than 0.05 were considered to be statistically significant.

The mean body weights of the five experimental groups were similar at the start of the experiment. After the treatment of 56 days, the body weights of all experimental groups were significantly decreased compared to the control group (Figure 1). NT-treated groups and the d-fenfluramine (d-FF) treated group lost weight during the first 48 hours of treatment. After this time, the weight gain of the d-fenfluramine (d-FF) group paralleled that of the control group. By contrast, the NT-treated groups continued to gain weight at a slower rate than the control group. It should also be noted that the rats that were pair fed to the high dose NT group (NT-H) gained weight at the same rate as the NT-H group (Figure 2). On day 56 of the treatment, the rats administered low dose NT (NT-L) and high dose NT (NT-H) had gained 24.6% and 33.2% less weight than the control group respectively. The rats administered d-fenfluramine (d-FF) gained 12.3% less weight than the control group. After termination of treatment at 56 days, there was no increase in the rate of weight gain of the NT treated group (NT-H-R) over the next 14 days and their weight difference to the control group (Control-R) was maintained (Figure 1).

The food intake of each group over the experimental period is illustrated in Figures 2 and 3. The rats in the control group consumed significantly more total food than the rats in any of the treatment groups over the 56-day period. The rats administered d-fenfluramine (d-FF) consumed less total food over the 56 day period than the control group. On the other hand, they consumed significantly more food than those in other groups, namely NT-H, NT-L and Pr-fed. The pair-fed rats ate slightly less than the NT-H group during the experiment, presumably due to spillage of the diet.

In this study, feed efficiency is expressed as weight gain (g) divided by weight of food consumed (Figure 4). The feed efficiency of the d-FF group, which was still higher than those of the NT-H, NT-L and Pr-fed groups, decreased significantly compared to the control group. The feed efficiencies of the NT-L and NT-H groups were 15.6% and 22.5% lower than that of the control group respectively. The feed efficiency of the d-FF group was 7.8% lower than that of the control group. Body weight gain and food intake were closely correlated in individual experimental groups (R² and P values for each group respectively: Control 0.719, P < 0.05; NT-H 0.907, P < 0.01; NT-L 0.796, P < 0.05; Pr-fed 0.694, P < 0.05; d-FF 0.798, P < 0.05) and in the combined data (Figure 5).

There were no significant differences in the weights of the kidney, spleen, liver, and gastrocnemius muscle among the groups after the 56-day experimental period (data not shown). The hearts of the rats in the control group were significantly larger than those in all other groups. However, when the weight of the heart was taken as a percentage of the body weight of the respective rat, there were no significant differences between the groups.

Parametrial and retroperitoneal white fat pads were significantly smaller in the NT-H group. They were also smaller in the NT-L and the Pr-fed groups but were not significantly different from either the NT-H group or the control group (Figure 6). There were no differences between the sizes of these adipose depots between the d-FF group and the control group. In the rats allowed to recover for two weeks after the end of treatment, the adipose depots remained smaller than those in the control group despite the fact that fat deposition increased significantly during the 2-week recovery period. The size of the inter-scapular brown adipose tissue depot was significantly reduced in all experimental groups compared to the control group (Figure 6).

The percentage of total body fat was significantly reduced in the NT-H group and a clear dose-related reduction with NT treatment was observed (Figure 7), while body fat was not significantly altered in any other experimental groups. The percentage of body water, as expected, increased slightly in the NT groups. There were no significant effects of any treatment on body protein or ash (i.e. mineral content). Changes in body composition during the treatment period could not be calculated because no rats were sacrificed for analysis at time zero. However, from percentage compositions and final body weights, we calculated the differences in the body composition between the treatment groups and control group at the end of the experiment. Virtually all the differences in body weight between the NT-H group and control group can be attributed to the difference in body fat between these two groups (Figure 8).

Serum leptin was reduced significantly in all the treatment groups compared to the control group (Figure 9). The differences in leptin values continued during the 14-day recovery period despite the withdrawal of NT, which might be due to a modest weight gain during that period. Serum leptin levels were correlated with percentage body fat for each individual experimental group (R² and P values for each group: Control 0.887, P < 0.001; Control-R 0.627, P < 0.05; NT-H 0.855, P < 0.01; NT-H-R 0.99, P < 0.001; NT-L 0.933, P < 0.001; Pr-fed 0.875, P < 0.001; d-FF 0.769, P < 0.01) and the combined data (R² = 0.708, P < 0.001) (Figure 10).

Serum prepared from trunk blood obtained at time of sacrifice (day 56) was used for clinical chemistry assays. The analyses included quantification of glucose, triglycerides, cholesterol, alkaline phosphatase, alanine aminotransferase, amylase, aspartate aminotransferase, chloride, cholesterol, creatinine phosphokinase, γ-glutamyl transpeptidase, glucose, hemoglobin, hematocrit, mean cell volume, platelets, potassium, sodium, triglycerides and white blood cells. One-way ANOVA plus Scheffes' Comparison of Means for each variable indicated that there were no significant differences between any of the serum analysis variables from the treatment groups and control group, nor among the treatment groups.

A small degree of alopecia during the first week of treatment was observed around the chins of the rats in the NT-H group. The alopecia disappeared within the next seven days after the animal technician was advised to be more scrupulous with the use of the intubation tube.

Rats in the NT treatment groups had yellowish urine which stained fur around the genitalia. Urine was tested negative for bilirubin. Rats in both NT-H and NT-L groups had loose stools for the first few days. By the end of the first week, the stools of the rats in the NT-L group were well formed and their consistency appeared normal, whereas the consistency of the stools of the rats in the NT-H group still appeared softer than those in other groups. Soft stools were observed among the rats in the NT-H group from time to time during the course of study. Moreover, the stools of the rats in both the NT-H and NT-L groups appeared reddish throughout the duration of the experiment. A test for hemoglobin was negative. The rats in the NT-H group liked to rub and groom the fur around their mouths after intubation, which was not observed in other groups. During the recovery period when the NT treatment was withdrawn, the color of the urine and stools returned to normal and the rats stopped grooming around the mouth. No other differences in activity/lethargy, fur condition, skin condition, eye condition or other physical characteristics were observed.