Combined Effects of Exercise and Phytoanabolic Extracts in Castrated Male and Female Mice

Abstract

Dry extracts from the Eurasian plants, Ajuga turkestanica, Eurycoma longifolia, and Urtica dioica have been used as anabolic supplements, despite the limited scientific data on these effects. To assess their actions on early sarcopenia signs, male and female castrated mice were supplemented with lyophilized extracts of the three plants, isolated or in association (named TLU), and submitted to resistance exercise. Ovariectomy (OVX) led to body weight increase and non-high-density cholesterol (HDL) cholesterol elevation, which had been restored by exercise plus U. dioica extract, or by exercise and TLU, respectively. Orchiectomy (ORX) caused skeletal muscle weight loss, accompanied by increased adiposity, being the latter parameter reduced by exercise plus E. longifolia or U. dioica extracts. General physical activity was improved by exercise plus herbal extracts in either OVX or ORX animals. Exercise combined with TLU improved resistance to fatigue in OVX animals, though A. turkestanica enhanced the grip strength in ORX mice. E. longifolia or TLU also reduced the ladder climbing time in ORX mice. Resistance exercise plus herbal extracts partly altered gastrocnemius fiber size frequencies in OVX or ORX mice. We provide novel data that tested ergogenic extracts, when combined with resistance exercise, improved early sarcopenia alterations in castrated male and female mice.

Keywords: Ajuga turkestanica; Eurycoma longifolia; Urtica dioica; aging; ergogenic phytotherapics; mice; orchiectomy; ovariectomy; resistance exercise; sarcopenia.

Figure 1

Identification of main active compounds in the ergogenic extracts. Cholesterol 50 mg/L (A) by LC-UV in 240 nm, (B) in 254 nm; Turkesterone (C) by LC-UV in 254 nm and (D) by LC-MS/MS with the fragments m/z 497.0 > 443.0 (light blue) and 497.0 > 461.0 (orange); Eurycomanone (E) by LC-UV in 254 nm and (F) by LC-MS/MS with the fragments m/z 391.5 > 251.1 (light blue) and 391.5 > 279.1 (orange); β-sitosterol (G) by LC-UV in 240 nm and (H) by LC-MS/MS with the fragments m/z 397.0 > 81.0 (light blue) and 397.0 > 95.0 (orange).

Figure 2

Effects of castration on uterus (A) and testes weight (C), 16 weeks after ovariectomy and orchiectomy, respectively, in comparison with sham-operated mice. Impacts of ovariectomy (B) and orchiectomy (D) on the body weight, 7 weeks after castration and before exercise and treatment onset. SHAM, sham-operated; OVX, ovariectomized; ORX, orchiectomized; Sed, sedentary; Exer, Exercise; Turk, Ajuga turkestanica; Long, Eurycoma longifolia; Urt, Urtica dioica; TLU, the association of the three extracts. Results are the mean ± SEM of 10 to 12 animals per group. ^a p < 0.05 significantly different compared to the sham-operated group (One-way ANOVA followed by Sidak post hoc test).

Figure 3

Changes in body weight in female (A) and male (D) mice, with treatments and exercise since castration until euthanasia. Area Under the Curve for the treatments and exercise period, from 8 to 16 weeks in females (B) and males (E). Final body weight of female (C) and male (F) mice. SHAM, sham-operated; OVX, ovariectomized; ORX, orchiectomized; Sed, sedentary; Exer, Exercise; Turk, Ajuga turkestanica; Long, Eurycoma longifolia; Urt, Urtica dioica; TLU, the association of the three extracts. Results are the mean ± SEM of 10 to 12 animals per group. ^a p < 0.05 significantly different compared with the sham-operated group treated with saline. ^b p < 0.05 significantly different compared with the castrated group treated with saline. ^c p < 0.05 significantly different comparing exercise to the respective sedentary group (One-way ANOVA followed by Sidak post hoc test).

Figure 4

Grip strength assessed at 12 and 16 weeks in female ((A) and (B), respectively) and male ((C) and (D), respectively) mice. SHAM, sham-operated; OVX, ovariectomized; ORX, orchiectomized; Sed, sedentary; Exer, Exercise; Turk, Ajuga turkestanica; Long, Eurycoma longifolia; Urt, Urtica dioica; TLU, the association of the three extracts. Results are the mean ± SEM of 10 to 12 animals per group. ^a p < 0.05 significantly different compared with the sham-operated group. ^b p < 0.05 significantly different compared exercise to respective sedentary treatment (two-way ANOVA followed by Uncorrected Fisher’s LSD).

Figure 5

Total time for ladder climbing (s) assessed at 16 weeks, in females (A) and males (B). SHAM, sham-operated; OVX, ovariectomized; ORX, orchiectomized; Exer, Exercise; Turk, Ajuga turkestanica; Long, Eurycoma longifolia; Urt, Urtica dioica; TLU, the association of the three extracts. Results are the mean ± SEM of 10 to 12 animals per group. ^a p < 0.05 significantly different compared with the sham-operated group treated with saline. ^b p < 0.05 significantly different compared with the operated group treated with saline (one-way ANOVA followed by Sidak post hoc test).

Figure 6

Assessment of spontaneous locomotor activity measured as the travelled distance for 5 min, at 12 and 16 weeks after castration in female ((A) and (B), respectively) and male mice ((D) and (E), respectively). Time-course for the travelled distance measured at three different periods: before castration, 12 and 16 weeks after castration, in female (C) and male mice (F). SHAM, sham-operated; OVX, ovariectomized; ORX, orchiectomized; Sed, sedentary; Exer, Exercise; Turk, Ajuga turkestanica; Long, Eurycoma longifolia; Urt, Urtica dioica; TLU, the association of the three extracts. Results are the mean ± SEM of 10 to 12 animals per group. ^a p < 0.05 significantly different compared with the sham-operated group. ^b p < 0.05 significantly different compared with the castrated group (two-way ANOVA followed by Uncorrected Fisher’s LSD).

Figure 7

Assessment of spontaneous locomotor activity measured as the animal speed for 5 min, at 12 and 16 weeks after castration in female ((A) and (B), respectively) and male mice ((C) and (D), respectively). SHAM, sham-operated; OVX, ovariectomized; ORX, orchiectomized; Sed, sedentary; Exer, Exercise; Turk, Ajuga turkestanica; Long, Eurycoma longifolia; Urt, Urtica dioica; TLU, the association of the three extracts. Results are the mean ± SEM of 10 to 12 animals per group. ^a p < 0.05 significantly different compared with the sham-operated group. ^b p < 0.05 significantly different compared to the operated group (two-way ANOVA followed by Uncorrected Fisher’s LSD).

Figure 8

Average of gastrocnemius cross-sectional area of female (A) and male mice (C) castrated and sham-operated in the different treatment groups. The gastrocnemius fiber size frequency distribution of female (B) and male mice (D) castrated and sham-operated in the different treatment groups. SHAM, sham-operated; OVX, ovariectomized; ORX, orchiectomized; Sed, sedentary; Exer, Exercise; Turk, Ajuga turkestanica; Long, Eurycoma longifolia; Urt, Urtica dioica; TLU, the association of the three extracts. Results are the mean ± SEM of 5 animals per group. ^a p < 0.05 significantly different compared to respective sedentary treatment. ^b p < 0.05 when comparing the groups in each area range. (One-way ANOVA followed by Sidak post hoc test.)

Figure 9

Representative histological images of gastrocnemius of sham-operated mice, sedentary or submitted to exercise, treated with saline (A,F), Turk 50 mg/kg (B,G), Long 200 mg/kg (C,H), Urt 50 mg/kg (D,I) or the combination of the three extracts TLU (E,J). Representative histological images of gastrocnemius of ovariectomized mice, sedentary or submitted to exercise, treated with saline (K,P), Turk 50 mg/kg (L,Q), Long 200 mg/kg (M,R), Urt 50 mg/kg (N,S) and combination of three extracts TLU (O,T). White arrow indicates gastrocnemius muscle fiber and black arrow indicates gastrocnemius muscle fiber in the group that showed a significant difference of cross-sectional areas (CSA). Turk, Ajuga turkestanica; Long, Eurycoma longifolia; Urt, Urtica dioica; TLU, the association of the three extracts.

Figure 10

Representative histological images of gastrocnemius of sham-operated mice, sedentary or submitted to exercise, treated with saline (A,F), Turk 50 mg/kg (B,G), Long 200 mg/kg (C,H), Urt 50 mg/kg (D,I) or the combination of the three extracts TLU (E,J). Representative histological images of gastrocnemius of orchiectomized mice, sedentary or submitted to exercise, treated with saline (K,P), Turk 50 mg/kg (L,Q), Long 200 mg/kg (M,R), Urt 50 mg/kg (N,S) and combination of three extracts TLU (O,T). Black arrow indicates gastrocnemius muscle fiber. Turk, Ajuga turkestanica; Long, Eurycoma longifolia; Urt, Urtica dioica; TLU, the association of the three extracts.

Figure 11

Average adipocyte cross-sectional area from inguinal white adipose tissue of female (A) and male mice (C) castrated or sham-operated in the different treatment groups. Adipocyte relative frequency area from inguinal white adipose tissue of female (B) and male mice (D), castrated and sham-operated, in the different treatment groups. SHAM, sham-operated; OVX, ovariectomized; ORX, orchiectomized; Sed, sedentary; Exer, Exercise; Turk, Ajuga turkestanica; Long, Eurycoma longifolia; Urt, Urtica dioica; TLU, the association of the three extracts. Results are the mean ± SEM of n = 5 to 7 animals per group (One-way ANOVA followed by Sidak post hoc test).

Figure 12

Representative histological images of adipocytes of inguinal white adipose tissue of sham-operated mice, sedentary or submitted to exercise, treated with saline (A,F), Turk 50 mg/kg (B,G), Long 200 mg/kg (C,H), Urt 50 mg/kg (D,I) or the combination of the three extracts TLU (E,J). Representative histological images of adipocytes inguinal white adipose tissue of ovariectomized mice, sedentary or submitted to exercise, treated with saline (K,P), Turk 50 mg/kg (L,Q), Long 200 mg/kg (M,R), Urt 50 mg/kg (N,S) and combination of three extracts TLU (O,T). Black arrow indicates adipocyte cell. Turk, Ajuga turkestanica; Long, Eurycoma longifolia; Urt, Urtica dioica; TLU, the association of the three extracts.

Figure 13

Representative histological images of adipocytes inguinal white adipose tissue of sham-operated mice, sedentary or submitted to exercise, treated with saline (A,F), Turk 50 mg/kg (B,G), Long 200 mg/kg (C,H), Urt 50 mg/kg (D,I) or the combination of the three extracts TLU (E,J). Representative histological images of adipocytes inguinal white adipose tissue of orchiectomized mice, sedentary or submitted to exercise, treated with saline (K,P), Turk 50 mg/kg (L,Q), Long 200 mg/kg (M,R), Urt 50 mg/kg (N,S) and combination of three extracts TLU (O,T). Black arrow indicates adipocyte cell. Turk, Ajuga turkestanica; Long, Eurycoma longifolia; Urt, Urtica dioica; TLU, the association of the three extracts.

Figure 14

Scheme representing the body weight variation and the summed weight of the three muscles (gastrocnemius, tibial, and soleus) when the animals were submitted to the exercise protocol, in the different treatment groups with: saline, Turk, Long, Urt and TLU, in female sham-operated and ovariectomized mice (A) and male sham-operated and orchiectomized mice (B). SHAM, sham-operated; OVX, ovariectomized; ORX, orchiectomized; Sed, sedentary; Exer, Exercise; Turk, Ajuga turkestanica; Long, Eurycoma longifolia; Urt, Urtica dioica; TLU, the association of the three extracts.