Adaptogenic Ashwagandha root extract modulates inflammatory markers in feline stress management: a double-blind placebo-controlled clinical trial

Figures & data

Figure 1. The schematic diagram of the clinical trial design to study the effect of ARE on stress in cats. ARE: Ashwagandha root extract.

Table 1. The schedule of different types of stress induced in cats.

S. No.	Week	Induction of Stress
1	Week – I Separation induced and new attendant at the clinic induced stress	(a) The cats were subjected to separation stress by isolating them from their regular companion and keeping them in isolated rooms. The cats were isolated for about 2 h. (b) Relocation of cat cages to another area of animal house and the animal keepers not talking or petting the cats all the day. (c) Introduction of multiple unfamiliar caretakers to conduct routine husbandry procedures, inconsistent husbandry schedule and withdrawal of play time
2.	Week – II Noise induced stress	(a) The cats were subjected to noise stress by bursting the firecrackers for 3 min. The normal range of cat’s hearing range for sounds is at a level of 80–90 decibels of sound. The firecrackers created a noise level above 105 dB in the animal room. It made all the experimental cats under severe stress. (b) The animal attendant come in and out of the animal house with intermittent banging the cage to made the cats under stress. (c) Playing a booming sound for 5 min using audio system made the cats under stress.
3	Week – III Restraining of cats	(a) Daily manipulation was carried out on all the cats. Each manipulation in a harsh manner was employed 3 times during the experimental period in both control and treatment groups. (b) Restrained the cat in a gunny bag and placed them in dark room for 20 min. (c) Frequent grabbing and holding the cat in hands for 10 min every time with gloved hands.
4.	Week – IV Travel and modification of feeding schedule induced stress	(a) All the cats were exposed to travel stress by placed into a cage and transported in a car for 20 mins. (b) Transfer of cats to different cages in the same animal room and cover the front side of the cage with black cloth for 12 h (6:00 AM to 6:00 PM). (c) The feeding time was delayed by 2 h and then the cats were fed. (d) Feed deprivation for 6–18 h for 1 time.

Figure 2. Effect of ARE interventions on important blood parameters, (A) Red blood cell count (RBC), (B) White blood cell count (WBC), (C) Mean corpuscular volume (MCV), (D) Mean corpuscular hemoglobin (MCH), (E) Platelet count, (F) Hemoglobin (Hb). Data n = 8; statistically analysed by Mean ± SEM. **Significantly different from placebo group at p < 0.01, ***Significantly different from placebo group at p < 0.001. ARE: Ashwagandha root extract.

Figure 3. Effect of ARE treatment on the biochemical markers of liver function. (A) ALT, (B) AST, (C) ALP, (D) Protein. Data n = 8; statistically analysed by Mean ± SEM. *Significantly different from placebo group at p < 0.05. ALT: Alanine transaminase, ALP: Alkaline phosphatase, ARE: Ashwagandha root extract, AST: Aspartate aminotransferase.

Figure 4. Effect of ARE treatment on important serum biochemical markers. (A) Serum albumin, (B) Serum globulin, (C) Serum creatinine, (D) Blood urea nitrogen (BUN), (E) Glucose. Data n = 8; statistically analysed by Mean ± SEM. **Significantly different from placebo group at p < 0.01, ***Significantly different from placebo group at p < 0.001. ARE: Ashwagandha root extract.

Figure 5. Effect of ARE treatment on oxidative stress markers. (A) Malondialdehyde (MDA), (B) Superoxide dismutase (SOD), (C) Glutathione (GSH), (D) Catalase. Data n = 8; statistically analysed by Mean ± SEM. **Significantly different from placebo group at p < 0.01, ***Significantly different from placebo group at p < 0.001. ARE: Ashwagandha root extract.

Figure 6. Effect of ARE treatment on cortisol and cytokines levels. (A) Cortisol, (B) Interferon-γ (IFN-γ), (C) Tumor necrosis factor-α (TNF-α), (D) Interleukin-10 (IL-10). Data n = 8; statistically analysed by Mean ± SEM. ***Significantly different from placebo group at p < 0.001. ARE: Ashwagandha root extract.

Figure 7. Effect of ARE treatment on important transcription factors viz; Nrf-2 and NFκB. (A) Nrf-2, (B) NFκB. Data n = 8; statistically analysed by Mean ± SEM. ARE: Ashwagandha root extract; Nrf-2: nuclear factor erythroid 2-related factor 2 (Nrf-2); NFκB: nuclear factor kappa light chain enhancer of activated B cells. Data n = 8; statistically analysed by Mean ± SEM. ***Significantly different from placebo group at p < 0.001. ARE: Ashwagandha root extract.