Streblus asper attenuates stress-induced physical and Biochemical changes in Rat Model


Sachin Neekhra1, Himani Awasthi1*, Dharmchand Prasad Singh2

1Amity Institutes of Pharmacy, Amity University Uttar Pradesh, Lucknow.

2College of Pharmacy, SR Group of Institutions, Jhansi.

*Corresponding Author E-mail:



The study deals with the evaluation of Streblus asper hydroalcoholic extract for adaptogenic activity using swimming endurance test and milk induced leukocytosis. The Streblus asper hydroalcoholic extract was administered to control vehicle, extract-treated (200 and 400 mg/kg p.o.) and standard drug (diazepam) treated groups prior to 1 hour of stress methods for 7 consecutive days. Mean swimming time, brain tissue antioxidant profiles (MDA, SOD, GSH, and Catalase), triglyceride, creatinine kinase, and corticosterone were evaluation parameters of adaptogenic activity in the swimming endurance test. However, determination of difference in number of leukocyte count considered as evaluation parameter was using milk induced leukocytosis method. The dose 400 mg/kg p.o. of Streblus asper hydroalcoholic extract for adaptogenic activity in both stress method showed significant variation (P< 0.1) when is compared with the stress control group. Therefore, it was revealed that Streblus asper hydroalcoholic extract showed potential adaptogenic activity.


KEYWORDS: Corticosterone, Creatinine kinase, Mean swimming time, Streblus asper, triglyceride.




Stress is threated homeostatic condition of the human being, and it is characterized in a form of non-specific response of the body1. Stress carries numerous physiological alterations in the living system, but a number of mechanisms of the body counteract to keep up homeostasis2. It was revealed that stress causes various diseases and disorders like hyperglycemia, raised blood pressure, gastric ulcers, and chronic depression3. The immune function is suppressed and the corticosterone level is elevated during stress situation which accelerates the formation of free radicals4-5. Therapy of stress includes alternative system of medicine, allopathic which is being currently available, but allopathic medicine drugs especially benzodiazepines category is having various adverse effect (side effect, tolerance, and dependence) which limit its use. Thus herbal formulation needs for management of stress on long term use6. Rasayanas of ayurveda are effective adaptogenic agents because they seem to prolong, Selye’s proposed stage of the “General adaptation syndrome”, the stage of resistance to stress, and prevent the final and third stage of exhaustion7.


On the current scenario, we need safer and economic herbal drugs as adaptogenic agents that can withstand stress without modifying the biological functions of the body. Plant Adaptogens like Withania somnifera8, Elutherococcus senticosus9, Panax ginseng10, Bacopa monniera11, Sidacordifolia12, Ocimum sanctum13, Butea frondosa14, Hypericum perforatum15 were reported. Streblus asper is a gnarled tree known by several common names mostly Sihor, Siamese rough bush. It is geographically present in the drier portion of India, Malaysia, Thailand, and Sri Lanka. S. asper contains myricetin, naringenin, kaempferol, quercetin, and ginkgetin flavonoids16-17. Some of the discovered flavonoid derivatives with a flavone-like structure such as quercetin and kaempferol have been therapeutically reported for adaptogenic activity. Therefore, we attempt to investigate the adaptogenic activity of S. asper hydroalcoholic extract using swimming endurance test and milk induced leukocytosis in rodents.




All conditions were maintained according to the CPCSEA guideline. The animals of either sex were selected randomly from animal house having uniform weight of 160±20 g. The room temperature was kept at 22±2°C with free access to water and food. One hour before the beginning of the experiment, animals were shifted to the laboratory and food and water were removed. The study protocol was approved by the institutional animal ethical committee (Approve Ref No. SRGI/COP/A/29/2016, CPCSEA Reg No. 1624/PO/a/CPCSEA).


Plant material:

The leaves of S. asper collected from forest region of Lakhimpur Kheri district, Uttar Pradesh, India, were authenticated by taxonomist of CSIR-NBRI, Lucknow, India. The voucher specimen of the plant was deposited in herbarium for future reference (NBRI/CIF/526/2016).


Preparation of extraction:

The powdered extract (S. asper) was taken and subjected to solvent extraction. The extraction of S. asper was carried out for 16 h with the hydroalcoholic solvent. A drug and solvent ratio 1: 5 w/v was maintained.


The S. asper hydroalcoholic extract was concentrated by evaporation of the solvent and continue it until dry at low temperature, then weighed, and calculated the percentage of different extractive values.


Phytochemical screening:

The hydroalcoholic extract of S. asper leaves was tested for various classes of chemical constituents by phytochemical tests for alkaloids, glycosides, flavonoids, phenolic compounds, saponins, and carbohydrates18.


Administration of the extracts:

Streblus asper hydroalcoholic extract was dissolved in dimethyl sulfoxide (DMSO) and diluted with distilled water. It was not exceed 3% of final concentration of DMSO solution. The diazepam drug was suspended in 2% gum acacia, administrated intraperitoneally (i.p.) before 30 min of exposure of stress. Extract DMSO solution of two different dose level 200 mg/kg and 400 mg/kg and vehicle DMSO solution as blank were administrated 1hr prior to exposure of stress.


Experimental design:

Twenty-five rats (five rats in each group) were randomly divided into five different groups. Group I consisted of unstressed rats received vehicle control; group II consisted of stressed rats those subjected to the forced swim stress for 7 days received vehicle control; group III and IV consisted of stressed rat received Streblus asper hydroalcoholic extract (200 and 400 mg/kg) and group V consisted of stressed rat received standard drug diazepam. (1 mg/kg)


Swimming endurance test in rats:

Stress was produced by force to swimming in cylindrical container (length 48 cm and width 30 cm) filled with water to a height of 25 cm till exhausted which was showed as drowning. The total swimming time for individual rat was recorded. Extracts were given to rats once daily for a period of 7 days before subjected to forced swim stress and on 8th day mean swimming time for each rat was calculated, the rat was anesthetized under the desiccator using ether and blood was collected through retro orbital sinus to estimate biochemical parameters like triglycerides19, corticosterone20-21, creatinine kinase22-23. The brain was dissection out from body and rinsed with ice cold normal saline followed by 0.15 M tris HCl (pH 7.4) for estimation of tissue antioxidant profiles24-27(MDA, SOD, GSH, and Catalase).


Milk-induced leucocytosis:

Leukocyte counts are increases after subcutaneous administration of pasteurized milk acts as an antigen which induces leucocytosis. It revealed infection like condition in which the efficacy of the drug determined to prevent leucocytosis similar to the stress-induced increase in leukocyte count.


Animals were divided into five groups having five animals in each group, and they received vehicle, S. asper hydroalcoholic extract with two doses of 200 mg/kg and 400 mg/kg, and reference drug diazepam (1.0 mg/kg) 1 h prior to the injection of milk 4 ml/kg sc. Twenty-four hours later, blood sample was collected from the retro orbital plexus and the leukocyte count was computed using Neubauers chamber28-29.


Statistical analysis:

All the values are expressed as mean ± SEM. Statistical differences between means were determined by one-way ANOVA followed by Dunnett’s post hoc test. P <0.05 was considered as significant. The statistical analysis was done using Instat® software (Graph pad Inc., Santabarba, CA)



Preliminary phytochemical investigation:

The preliminary phytochemical screening with leave of Streblus asper L showed the presence of glycosides, alkaloids, flavonoids, phenolic compounds, saponins, and carbohydrates in hydroalcoholic extract.


On 8th day animals in stress control group swam for 203±2.7Sec and with standard and hydroalcoholic extracts treated showed a significant (p<0.05) increase in swimming endurance time (Table 1).

Table 1: Effect of S.asper hydroalcoholic extract on mean swimming time in rats

S. No.

Treatment group

Mean swimming time (S)


Normal Control (10 ml/kg)



Stress Control (10 ml/kg)



S. asper hydroalcoholic extract (200 mg/kg)



S. asper hydroalcoholic extract (400 mg/kg)



Standard drug diazepam (1 mg/kg)


Value are expressed as mean± SEM (n = 5), one way ANOVA followed by Dunnett's test; ###P<0.001 when compared to normal control *P <0.5, **P < 0.1, ***P < 0.05 when compared with the stress group


Table 2: Effect S. asper hydroalcoholic extract on biochemical parameters in rats

S. No.

Treatment group





Creatine Kinase



Normal Control (10 ml/kg)





Stress Control (10 ml/kg)





S. asper hydroalcoholic extract (200 mg/kg)





S. asper hydroalcoholic extract (400 mg/kg)





Standard drug diazepam (1 mg/kg)




Value are expressed as mean± SEM (n = 5), one way ANOVA followed by Dunnett's test; ###P<0.001 when compared to normal control * P <0.5, **P < 0.1, ***P < 0.05 when compared with the stress group


Table 3: Effect of S. asper hydroalcoholic extract on antioxidant profiles in rats

S. No.

Treatment group










Normal Control (10 ml/kg)






Stress Control (10 ml/kg)






S. asper hydroalcoholic extract (200 mg/kg)






S. asper hydroalcoholic extract (400 mg/kg)






Standard diazepam (1 mg/kg)





Value are expressed as mean± SEM (n = 5), one way ANOVA followed by Dunnett's test; ###P<0.001 when compared to normal control * P <0.5, **P < 0.1, when compared with the stress group


Table 4: Effect of S. asper hydroalcoholic extract on total leukocyte count in rats

S. No.

Treatment group

Difference in no. of leukocyte (cu/mm)


Normal Control (10 ml/kg)



Stress Control (10 ml/kg)



S. asper hydroalcoholic extract (200 mg/kg)



S. asper hydroalcoholic extract (400 mg/kg)



Standard drug diazepam (1 mg/kg)


Expressed as mean± SEM (n = 5), one way ANOVA followed by Dunnett's test, ### P < 0.001, when compared with normal control, * P <0.5, **P < 0.1, ***P < 0.05 when compared with the stress group



Biochemical parameters like triglyceride, creatinine kinase and corticosterone were found to be increased in forced swimming stress model in rats which were significantly decreased (*P<0.5, **P < 0.1) on pretreatment with S. asper hydroalcoholic extract, (200 and 400 mg/kg) whereas diazepam (1 mg/kg) significantly decreased (***P< 0.05) reduced glucose, cholesterol and corticosterone (Table 2).


Other biochemical parameters like MDA, SOD, GSH and Catalase altered levels to be found in forced swimming stress model in rats. Increased level of MDA was significantly reduced (*P <0.5, **P < 0.1, ***P < 0.05) on pretreatment with S. asper hydroalcoholic extract (200 and 400 mg/kg) and diazepam (1 mg/kg), whereas decreased of SOD, GSH and Catalase were increased on pretreatment with S. asper hydroalcoholic extract (200 and 400 mg/kg) and diazepam (1 mg/kg) (Table 3).


Total leukocyte count was significantly reduced (*P <0.5, **P < 0.1, ***P < 0.05) on pretreatment with S. asper hydroalcoholic extract (200 and 400 mg/kg) and diazepam (1 mg/kg) after administration of pasteurized milk (Table 4).



Swimming endurance test is extensively used method among to those method used for antistress activity of drugs. When animals are subjected to swim in a water container, which is not their natural habitat, they initially show struggling and swimming activity to escape from the water. Later on they become immobile due to depression, which is a common response during   stress30-31.


As a result of this immobility, drowning and death of rat occurs. S. asper hydroalcoholic extract have significantly increased the survival time or swimming endurance of rat at the tested doses of 200 and 400 mg/kg when compared to the untreated stress rat. The dose of 400 mg/kg of S. asper hydroalcoholic extract has significant variation (P>0.1) when compared to the standard drug (diazepam 1mg/kg) as found to be increased swimming survival response of rat.


The target for different stressors is the brain, as it is more sensitive for degeneration due to stress32. It results in hypothalamic- pituitary axis (HPA) activation, leading to release of adrenocortical hormone responsible for stressful response further releasing of corticosterone hormone33. In forced swim stress model repeated exposure of rats for 7 days to stressful condition was carried, as it causes a wide range of physiological and neuroendocrine changes34.


During stress, hypothalamic-pituitary-adrenocortical axis activation leads to increase in corticosterone. The effect of stress on triglycerides was shown due to liberation of corticosterone from adrenal cortex that leads to mobilize lipids from adipose tissues.


Stress-induced elevate levels in plasma glucose and creatine kinase may be attributed to corticosterone-mediated enhanced metabolism to meet the increased demands of the body organs during stress35-36. The dose of 400 mg/kg of S. asper hydroalcoholic extract was significant reduced (P>0.1) triglycerides, creatine kinase and corticosterone level with compared to stress control.


Oxygen demand of brain increases during stress. The brain gets oxidative stress as results of oxidation and formation of free radicals occurs.


The balance between oxidants and antioxidants is generally validated using the swimming endurance test which exhibited as neuronal cell damage and death37.


In this experiment work, untreated rat allowing the swimming endurance test was found to exhibit elevate lipid peroxidation (MDA) and lower antioxidant enzyme SOD, GSH and Catalase levels.


The effect of S. asper hydroalcoholic extract against stress induced brain lipid peroxidation and alteration of level of SOD, GSH and Catalase was found to be dose dependent. S. asper hydroalcoholic extract at the dose of 400 mg/kg exhibited nearly similar effect to that of standard diazepam drug.


Milk-induced leukocytosis is novel model which recently used for evaluation of anti-stress activity. The after 24 hours of subcutaneous administration of pasteurized milk, markedly and significantly increased leukocyte count was observed38-39. As above results, the dose 400 mg/kg of S. asper hydroalcoholic extract has markedly affected on biochemical parameters. So, the dose of 400 mg/kg of S. asper hydroalcoholic extract was significantly reduced leukocyte count as showed effect produced by this plant due to abolishing of non-specific resistance of body. S. asper contains some bioflavonoids (kaempferol, myricetin, and ginkgetin) which widely present in laves throughout whole part of this plant40. In my research finding that bioflavonoid especiallyquercetin-3-O rutinoside or rutin presents in S. asper leads to modulate GABA receptor in brain. Therefore, S. asper will mark as adaptogenic agent category and served as therapeutic agent into clinical setting in future.



S. asper hydroalcoholic extract in 400 mg/kg dose had adaptogenic activity which characterized by forced swimming time, triglyceride, creatinine kinase, corticosterone levels in plasma as well as MDA, SOD, GSH and Catalase levels in brain tissue. However, effect of S. asper hydroalcoholic extract not yet restores as normal control result. Therefore, S. asper hydroalcoholic extract may provide protection against stress probably by preventing of biochemical alteration and humoral perturbations during stress and their adverse implications on body physiology. Thus S. asper hydroalcoholic extract may play a key role as complementary and alternative medicine in future for attenuation of stress induced biochemical alteration.



The author gratefully acknowledges work supported by Dr. DCP Singh, Director, College of Pharmacy, SR Group of Institutions, Jhansi.



There is no conflict of interest into the manuscript.



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Received on 23.02.2020            Modified on 30.05.2020

Accepted on 10.07.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(4):1910-1914.

DOI: 10.52711/0974-360X.2021.00337