INTRODUCTION:

Plants been used for the treatment of various ailments since prehistoric times, they are also called as medicinal herbs¹. As stated by WHO, in developing countries more than 80% of population uses herbal medicines for the treatment of commonly occurring diseases. India is being a country with enormous wealth of traditional plants having medicinal quality. WHO recognized herbal medicines as one of the necessary component of primary health system and almost 11% of drugs were formulated using herbal plants².

Flax seed scientifically known as Linum usitatisimum, L. is a specie of Linaceae family, it is relatively short plant being cultivated in cooler region of the world, its seeds are known as linseed, two varieties/ color of linseed occurs namely: yellow and brown, in which brown color seeds were more common and has good nutritional benefits. Seeds being a good source of essential fatty acids, digestible proteins and lignan³. Lignan are di-phenolic compounds they have estrogenic properties and has potential in prevention and treatment of cancer⁴. Essential fatty acids possess wide range of health benefits for human population, they must be incorporated into the diet as they are not synthesized into the human body⁵. Thus flax seed become an important functional food.

Depression and anxiety are two extremely common neurotic disorders affecting different age groups and genders globally. WHO reported in 2017 that around the world an estimated 322 million populations were affected by depression⁶. Many drugs has been developed for the treatment of depression like lithium salts, tricyclic antidepressant, monoamine oxidase inhibitors and serotonin inhibitors but they have side effects like sleeplessness, constipation, dry mouth and even impotency⁷. Thus natural products have attracted the attention of many researchers in formulating drugs with higher safety and lower cost. Naturally occurring PUFAsi. essential fatty acids intake in the diet can help in the management of neurotic disorders by influencing nervous system, altering neural activity and neurotransmitter synthesis⁸. Flax seed is one of the most common natural product on which researches being conducted but not much of the work was done using models of animalsforanxiety and depressionthus present study has objective to investigate the effects of repeated administration of flax seed extract in some behavioural changes in stress induced mice.

MATERIALS AND METHODS:

Material:

Flax seeds were obtained from local market of Noida, India. The material were cleaned many times with plain water and then dried in open air. The seeds which were dried was further vacuum-packed and stored at 180°C in the dark until they were used in tests.

Methods:

Preparation of extract:

The stored seeds were grounded into fine powder and extraction of 100gm seed was done using Soxhlet apparatus and as solvent ethanol is used at 70-90⁰C for 8-9h. Then evaporation of solvent was done at room temperature and extract was further stored at 4⁰C and used for analysis⁹.

Phytochemical analysis:

Preliminary freshly generated crude ethanolic extracts of were subjected to quantitative phytochemical analysis. Flax seeds (Linum usitatisimum, L.) to identify different phytoconstituents using the approach employed by Amin and Thakur, such as alkaloids, glycosides, tannins, flavonoids, proteins, terpenoids, phytosterols, and phenols¹⁰

DPPH technique for determining anti-oxidant properties:

Blois' technique was used to test the anti-oxidant properties of Flax seeds (Linum usitatisimum, L.)extract using 1, 1- diphenyl-2-picrylhydrazyl (DPPH)¹¹. In ethanol, 0.2mmol/l of DPPH was produced and used as a control solution. 500ml of the control solution was added to the extract solution of various concentrations (100-500g/ml). Each combination was thoroughly mixed before being set aside for 30minutes at room temperature. With the use of a UV-Visible spectrophotometer, the absorbance of each solution was measured at 517nm, and the concentrations were calculated to compare the absorbance of ascorbic acid under identical conditions¹². Equation was also used to calculate antioxidant activity 1.

I % = (Ac –As) / Ac X 100 ¾ equation 1

Where, I stands for inhibition, Ac for control absorbance, and As for extract absorbance

Standard drug:

Diazepam was referred as standard drug used for evaluating anti-anxiety activity in mice. Drugs were powdered and made into suspension in distilled water.

Animals:

The guidelines for animal studies are provided by the institution's ethical committee. Swiss albino mice of either sex, weighing between 25 and 30g, were housed in a group of six in polypropylene cages, exposed to standard laboratory conditions, including a 12hour cycle of darkness and light, a temperature of 22°C±2°C, and free access to drinking water. Standard laboratory pellet diet was also fed to the animals. The Institutional Animal Ethics Committee granted approval (Regn No. 1173/ac/po/08/CPCSEA) to use laboratory animals in accordance with the Committee for the Purpose of Control and Supervision of Experiments on Animals' (CPCSEA) standards for such experiments.

Protocol for the Experiment:

Animals were initially trained to ingest a 2% sucrose solution at the beginning of the trial. After tracking the relationship between sucrose intake and body weight for two weeks, groups were created. The study involved a total of 30 mice of each sex. Five distinct groups of mice (n = 6/group) were used to assess the anti-anxiety activity. Distilled water (10ml/kg), diazepam (5mg/kg), and incremental dosages of 40, 60, and 80mg of flax seed (Linum usitatisimum, L.) extract were given orally to the control, standard drug, and three test groups, respectively.

Groups for Experimental animals:

The following groups (n = 6) were used to examine the way an extract of flax seeds (Linum usitatisimum, L.) affected mice with models of anxiety and depression. One hour after the vehicle or medication was administered orally (p.o.) in the experimental test.

· Group 1 – Control: (Distilled water) (10ml/kg, p.o.) – [Control]

· Group 2 – Standard drug: Imipramine (20mg/kg, p.o)

· Group 3 – Flax seeds (Linum usitatisimum, L.) extract (40mg/kg, p.o.) ‑[FSE 40]

· Group 4 – Flax seeds (Linum usitatisimum, L.) extract (60 mg/kg, p.o.) ‑ [FSE 60]

· Group 5 – Flax seeds (Linum usitatisimum, L.) extract (80 mg/kg, p.o.) ‑ [FSE 80].

Chronic Mild Stress procedure:

The mice were subjected to the chronic stress protocol developed by Shivanna.et.alwith slight modifications¹³. Stress was given after 2 weeks of adaptation with 2% sucrose, except for the control group (Group I), all the animals were subjected to chronic mild stress, procedure was done as follows:

16h of water deprivation; 5h water and food deprivation prior to sucrose test (2days); 17h of overnight illumination; exposure to low temperature at 0°C for 5h, these stress procedure ran for 30 days.

Experimental models for depression:

Forced Swim Test (FST):

Porssolt created the forced swimming test to examine how animals' conduct changed after receiving antidepressants.(1978, Porsolt et al. Each animal was placed inside a vertical plexiglass cylinder that measured 40 cm in height and 18 cm in diameter and held water that was kept at a temperature of 24 to 25°C14 up to a height of 15cm. After the first two to three minutes of struggle in the water, mice typically settle into what is known as the immobility phase, where they remain floating in the water with little activity other than to maintain their heads above water. Each animal was submerged in the water for 6 minutes throughout the test. The final four minutes of the FST were marked by immobility time. Each animal was submerged in the water for 6 minutes throughout the test. The final four minutes of the FST were marked by immobility time. The mice were taken out of the water, allowed to dry for 15 minutes, and then put back inside their cage. The reduction in immobility time was associated with an antidepressant effect.

The Tail Suspension Test:

Steru. et. al. created the tail suspension test in 1985 as an alternative to the forced swim test for determining a substance's anti-depressive potential. With the aid of an adhesive tape that was put about 1 cm from the tip of the tail for this test, each mouse was suspended from the edge of a 58 cm high table top. Five minutes were tracked as the length of immobility. When a mouse hung passively and was still for at least one minute (apart from breathing), it was deemed to be immobile. The length of time that each group spent immobile was compared. A shorter period of immobility is a sign of an antidepressant's impact.¹⁵

Statistical Analysis:

The information in the table was shown as the replicate average SD. Using SPSS software (version 9.0), the data was statistically analysed by doing an analysis of variance (ANOVA). Turkey HSD was used to compare the significance of their means, and a p value of 0.05 was deemed significant.

RESULT:

Determination of phytochemicals:

According to the results of the phytochemical examination of the flax seed extract, glycosides, phytosterols, flavonoids, phenolic compounds, alkaloids, and amino acids were present (Table 1)

Table 1: Ethanolic extract of flax seeds (Linum usitatisimum, L.) preliminary phytochemical analysis

Phytoconstituents	Present/absent
Alkaloids	+
Phenols	+
Tannins	+
Flavonoids	+
Proteins/amino acids	+
Carbohydrates	-
Fats/oils	+
Sterols	-
Terpenoids	+
Glycosides	-

In- vitro Antioxidant activity:

DPPH (2,2'-diphenyl-1-picrylhydrazyl) radical scavenging activity was the most extensively used method for assessing antioxidant efficacy in plant and biological samples¹⁶. Table 2 shows that the ethanolic extract of Flax seeds (Linum usitatisimum, L.) extract had DPPH radical scavenging action in a concentration-dependent manner. The IC₅₀ value is the concentration required to inhibit DPPH by half. The IC₅₀ value of the ethanolic extract of flax seeds is 80g/ml, indicating that doses of 80 g/ml are required to lower DPPH by 50%.

Table 2: Inhibition percentage of DPPH radical by ethanolic extract and Ascorbic acid

Concentration of extract (µg/ml)	Percentage inhibition of DPPH radical
Concentration of extract (µg/ml)	Flax seeds	Ascorbic Acid
40	45.74± 0.46	52.56±0.40
60	53.32±0.60	55.73±0.44
80	58.65±0.46	60.26±0.66
100	66.70±0.44	67.04±0.83
200	70.23±0.29	71.39±1.03

Quantitative analysis of anti-oxidative components:

The Total phenolic content (TPC) of ethanolic extract of Flax seeds (Linum usitatisimum, L.) was estimated to be 37.460.10mg of Gallic acid equivalent (GAE) per gramme of extract using a standard calibration curve of Gallic acid. This score indicates that the seeds include a reasonable amount of phenolic chemicals, which can be used medicinally¹⁷.

Sucrose intake in mice:

Sucrose intake was observed to decrease from 1.98 ml to 0.84ml per day in mice exposed to mild stress. Normal control group maintained the sucrose intake compared to imipramine fed animals. Higher sucrose intake was observed in higher dosage of Flax seeds (Linum usitatisimum, L.) extracti.e 80mg/kg. (P<0.05), which was not, different from imipramine fed animals. Results were revealed in fig 1.

Fig 1: Consumption of sugar in mice under mild, ongoing stress

Effect of Flax seeds (Linum usitatisimum, L.)extraction the duration of immobility in FST and TST:

Flax seeds (Linum usitatisimum, L.) extract (40, 60 and 80mg/kg, p.o) decreased the immobility time in forced swim test [F (4,29) = 521.59, P<0.05] and tail suspension test (TST) [F(4,29)= 1168.63, P<0.05] as compared to control group. Imipramine, (20mg/kg, p.o.) used as a positive control, showed effects similar to Flax seeds (Linum usitatisimum, L.) extract 80mg/kg, p.o. which is statistically significant thus dose of 80mg/kg, p.o was the most effective dose in reducing mobility time in the forced swim test and this was chosen for subsequent experiments in the model of depression.

Table 3: Effect of different dosages of Flax seeds (Linum usitatisimum, L.)

Treatment	FST (s)	TST (s)
FSE 40	160.88^a±1.14	37.27^a±1.50
FSE 60	125.83^b±1.28	41.66^b±1.43
FSE 80	95.55^c±1.28	152.44^c±1.17
Control	162^d±1.34	136.44^d±1.70
Imipramine	92.16^c±2.56	156.38^c±2.92

Above table showed the effect of different dosages of Flax seeds (Linum usitatisimum, L.)extract (FSE40, FSE60 and FSE 80mg/ml) seed extract on duration of immobility in FST (significance at 0.05 level), mean value having different superscript letters for each element are significantly different, only FSE 80 and Imipramine has same superscript thus indicate no significant difference between them.

DISCUSSION:

Herbal plants from very ancient times used for many pharmacological preparations, depending on the formulation different parts of the plant like stems, leaves, flower and seeds were used as they are rich in phytochemicals such as tannins, glycosides, terpenoids, phenols, steroids, etc. natural antioxidants are present in seeds and progression of certain chronic and acute diseases have been reduced by their usage¹².

Preliminary phytochemical screening of flax seed extract confirmed the presence of various classes of secondary metabolites such as tannins, phenols, flavonoids,glycosides, carbohydrate, fats/oils.These are likely to be responsible for free radical scavenging activity of the extract. In the present study total phenolic content of the extract was found to be 37.46±0.10mg/g of GAE per gram of extract thus indicate that it is a rich source of polyphenols. Further for antioxidant activity DPPH method was used for evaluation of free radical scavenging property of the extract, DPPH is an organic stable free radical, antioxidant scavenged it through donation of hydrogen to form DPPH-H molecule which results in colour change from purple to yellow^16,17.

Antioxidant capacity of the extract was also expressed as IC₅₀which indicates the concentration required to reduce half of DPPH, the IC₅₀value of the extract is found to 299.98mg/ml which is an indicate that given concentration is required to scavenge half of DPPH.

Depression and anxiety are the most prevalent mental disorders which widely effect world’s population. Diagnosis, treatment and recovery of these disorders become a great challenge due to the complexities of nervous system which deliberately affect the whole process¹⁸. More of the contribution is required to reduce the global impact of these disorders. For the treatment of depression and anxiety serotonin- specific reuptake inhibitors (SSRIs), antidepressants and benzodiazepines have been prescribed to patients¹⁹. These classes of drugs were effective but have unwanted side effects. Hence, there is need to develop more effective and safer alternatives, thus this study focuses of effect of flax seed extract on treatment of depression and anxiety.

Brain functions are closely related to intake of energy from carbohydrate sources like sucrose.A much greater sucrose intake was observed in the flax seed extract (80mg/ml) and imipramine group. It has been shown that higher intake of polyphenols increases glucose intake, there by stimulating neurons for better cognitive function, indicating reduced oxidative stress.Fig 1 suggests that higher dosage of the extract can be supplemented for improved cognitive functions.

Behavioral changes were reported in animals exposed to stress, animal models were used for evaluating the efficacy anti-depressive and anti-anxiety properties of different natural formulations²⁰. In the present study anti-depressive and anti-anxiety properties of flax seed extract were studied using behavioral changes in mice.

Forced Swim test and elevated plus maze method was used to indicate the behavioral changes in mice, as result indicated flaxseed extract of high dosage i.e 80mg/kg p.o reduced the immobility time of mice when compared with imipramine. This effect is may be due to the presence of omega-3 fatty acids in the extract as these fatty acids modulates functioning of brain, regulates neuron receptors and also plays a role in managing oxidative stress of brain, they also have a potential in the treatment of various neuropsychiatric disorders²¹.

Similar results were seen for elevated plus maze method, thus the extract of higher dosage i.e 80 mg/kg body weight shows anti-depressive and anti-anxiety effect.

CONCLUSION:

In a summary, our finding clearly indicate that the effect of flaxseed extract on depression and anxiety was found to be effective but its dosage dependent, as concentration increases the effect also increases as compared to medication used in depression and anxiety, this effect is may be due to the presence of essential fatty acids i.e omega-3,poly phenols and lignans in the extract²². Furthermore, biochemical and molecular biological studies are needed to determine the exact mechanism by which theflaxseeds extract exhibit anti-depressive and anti-anxiety like effect.

CONFLICT OF INTEREST:

None.

REFERENCES:

1. Kasote DM. Jagtap SD. Thapa D.Khyade MS. Russell WR. Herbal remedies for urinary stones used in India and China: A review. Journal of Ethnopharmacology. 2017; 203: 55-68. https://doi.org/10.1016/j.jep.2017.03.038

2. Islam MT. Sarkar C. El‐Kersh DM.Jamaddar S. Uddin SJ. Shilpi JA. Mubarak MS. Natural products and their derivatives against coronavirus: A review of the non‐clinical and pre‐clinical data. Phytotherapy Research. 2020; 34(10): 2471-92. https://doi.org/10.1002/ptr.6700

3. Halligudi N. Pharmacological properties of flax seeds: a Review. Hygeia. JD Med. 2012; 4(2): 70-7. http://www.hygeiajournal.com/downloads/65504860670-77%20dr.pdf

4. Gupta R. Meghwal M. Prabhakar PK. Bioactive compounds of pigmented wheat (Triticum aestivum): Potential benefits in human health. Trends in food Science & Technology. 2021; 1;110:240-52.https://doi.org/10.1016/j.tifs.2021.02.003

5. Calder PC. Functional roles of fatty acids and their effects on human health. Journal of Parenteral and Enteral Nutrition. 2015;39:18S:32S. https://doi.org/10.1177/0148607115595980

6. Friedrich MJ. Depression is the leading cause of disability around the world. JAMA. 2017; 18(317(15): 1517 doi: 10.1001/jama.2017.3826.

7. Krishnan KR. Monoamine oxidase inhibitors. The American Psychiatric Association Publishing Textbook of Psychopharmacology. 2017;10;283.https://doi.org/10.1176/appi.books.9781615371624

8. Lange KW. Omega-3 fatty acids and mental health. Global Health Journal. 2020: 1; 4(1):18-30. https://doi.org/10.1016/j.glohj.2020.01.004

9. Aravind R.Aneesh TP. Bindu AR. Bindu K.Estimation of Phenolics and Evaluation of Antioxidant activity of Cinnamomum malabatrum (Burm.F). Blume. Asian J. Research Chem. 2012; 5(5): 628-632. Available on: https://ajrconline.org/AbstractView.aspx?PID=2012-5-5-13

10. Tawheed A. Monika T. A comparative study on proximate composition, phytochemical screening, antioxidant and antimicrobial activities of Linumusitatisimum L.(flaxseeds). International Journal of Current Microbiology and Applied Sciences. 2014; 3(4): 465-81.

11. Blois MS. Antioxidant determinations by the use of a stable free radical. Nature. 1958; 26; 181(4617): 1199-200.https://doi.org/10.1038/1811199a0

12. Kishu Tripathi. Herbs for Depression: A Review. Research J. Science and Tech. 2009; 1(2): 55-62Available on: https://rjstonline.com/AbstractView.aspx?PID=2009-1-2-2

13. Naveen S. Siddalingaswamy M. Singsit D. Khanum F. Anti‐depressive effect of polyphenols and omega‐3 fatty acid from pomegranate peel and flax seed in mice exposed to chronic mild stress. Psychiatry and Clinical Neurosciences. 2013; 67(7): 501-8.https://doi.org/10.1111/pcn.12100

14. Skolnick P. Layer R. Popik P. Nowak G. Paul I.TrullasRamon. Adaptation of N-Methyl-D-Aspartate (NMDA) Receptors following Antidepressant Treatment: Implications for the Pharmacotherapy of Depression. Pharmacopsychiatry.1996; 29: 23-6. doi: 10.1055/s-2007-979537.

15. Jin L. Wu F. Li X. Li H. Du C. Jiang Q. You J. Li S. Xu Y. Anti‐depressant Effects of Aqueous Extract from Acanthopanax senticosus in Mice. Phytotherapy Research. 2013; 27(12): 1829-33. https://doi.org/10.1002/ptr.4938

16. Sirivibulkovit K.Nouanthavong S.Sameenoi Y. Paper based DPPH assay for antioxidant activity analysis. Analytical sciences. 2018;34(7):795-800.https://doi.org/10.2116/analsci.18P014

17. Russo R. Reggiani R. Phenolics and antioxidant activity in flax varieties with different productive attitude. International Food Research Journal. 2015; 1; 22(4): 1736. http://www.ifrj.upm.edu.my

18. Mulder GB. Pritchett K. The elevated plus-maze. Journal of the American Association for Laboratory Animal Science. 2004 ; 43(2): 39-40.doi:10.1124/jpet.108.139261

19. Shaida B. Dubey AK. Singh K. Sharma P. Anti-depressive and Anti-anxiety Like Effects of Cydonia oblonga in Mice Exposed to Mild Stress. Indian Journal of Public Health. 2019; 10(10). doi:10.5958/0976-5506.2019. 02802.X

20. Miyazaki Y. Kobayashi K. Matsushita S. Shimizu N. Murata T. An assessment of the spontaneous locomotor activity of BALB/c mice. Journal of Pharmacological Sciences. 2022; 149(2): 46-52. https://doi.org/10.1016/j.jphs.2022.03.001

21. Kumara P. Sunil K. Arun Kumar B. Determination of DPPH Free Radical Scavenging Activity by RP-HPLC. Rapid SensitiveMethod for the Screening of Berry Fruit Juice Freeze Dried Extract. Nat Prod Chem Res. 2018; 6: 341.DOI: 10.4172/2329-6836.1000341

Received on 17.03.2022 Modified on 30.12.2022

Research J. Pharm. and Tech 2023; 16(11):5005-5009.

DOI: 10.52711/0974-360X.2023.00810