INTRODUCTION:

Anxiety is an emotional state, unpleasant in nature, associated with uneasiness, discomfort and concern or fear about some defined or undefined future threat. It is common disorder affecting around 15-20% of the total population and has become a very important area of research in psychopharmacology^1,2. All undergo anxiety at one or other stages of their life but the condition is soon balanced or reversed. It has been found that women are affected more when compared to men. Treatment is needed when the person is unable to balance the situation and the condition is worsened and excessive. Some psychotics and depressed patients also exhibit pathological anxiety.

Usually anxiety disorders are associated with other mental disorders such as drug abuse and depression. Individuals with anxiety disorders face excessive fear and worry which makes them to avoid many situations which might increase anxiety levels or worsen the control over the situation.^{3, 4} Many people feel anxiety in specific situations but the people suffering from this feeling will face unrealistic situations and tend to become socially inactive. Effective treatment such as anxiolytic drug therapy or cognitive-behavioural therapy exist, but many patients experience adverse effects or do not benefit from full symptom control. Benzodiazepines are usually used in the treatment of anxiety. However, the clinical uses of benzodiazepines are limited. A review on earlier studies conducted, revealed that Panchagavya products possess bioenhancing property, hepatoprotective activity, anticonvulsant activity etc⁵. In recent time it has been noted to be used in the cancer treatment and psychiatric disorders as well. Panchagavya ghrita is one of the broadly used ayurvedic formulation and it is described in both Ayurvedic Pharmacopeia of India (API) as well as in Ayurvedic formulary of India. Panchagavya ghrita is a formulation consisting of five ingredients Goksheera (cow milk), Godadhi (cow’s curd), Gomootra (cow’s urine) Gomaya (cow’s dung) and Goghrita (cow. ghee) obtained from the Indegenous cow Bos indicus. According to Ayurvedic formulary of India (IFA) Panchagavya ghrita is indicated in the treatment of Epilepsy (apasmara), fever (jawara), psychiatry diorders (unmada), Atattvabhinivesha (Obsessive compulsive disorder) and jaundice ( kamala)⁶ The present study was carried out with an objective of scientifically validating the traditional claims and to compare the ancient knowledge with the latest pharmacological aspects of preclinical research. Further there were no scientific data available regarding the antianxiety activity of Panchagavya Ghrita. Hence the present study was under taken.

MATERIALS AND METHODS:

Panchagavya ghrita formulation composed of cow milk, curd, ghee; urine & dung from Indigenous cows (Bos indicus) were obtained as and when required from the Goushalas belonging to Sri Ramachandrapura Math, Hosanagara, Karnataka.

Selection of animals:

Mice of either sex, 4-6 weeks, weighing (25-30g) were obtained from central animal house, NUCARE, Paneer, Mangalore. The animals were appropriately grouped and then sheltered in distinct cages. The cages were kept under standard lab conditions of temperature 25 ± 2° with appropriate dark and light cycle of 12 hours. Free access to standard food and water ad libitum was permitted for the animals. The investigation was done in accordance to the guidelines of the CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals), New Delhi, India.The research work was permitted and approved by the Institutional Animal Ethics Committee (NGSMIPS/IAEC/MArch-2018/52.)

Selection of dose⁷

The Animal dose of Panchagavya Ghrita was calculated from Human dose. In Ayurveda system of medicine, the dose of Panchagavya Ghrita is 3 Karsa to1 pala (1 Karsa = 12g, 1 Pala = 48g.) i.e. 36 & 48g of the preparation. This Human dose was converted into mice dose by multiplying with a factor of 0.0026×50. Hence the dose of Panchagavya Ghrita for evaluating anti-depressant activity using mice was 4680 mg/kg (36×.0026×50) and 6240 mg/kg (48×.0026×50).

Experimental design:

The anti-anxiety activity Panchagavya Ghrita was evaluated on experimental animals by using three models i.e Elevated plus maze model, light -dark transition model and open field test models using two dose levels i.e. 4680 mg/kg and 6240 mg/kg.

Treatment schedule

Group	Treatment
Group I	Normal control (vehicle 10ml/kg) p.o
Group II	Standard drug (Diazepam, 2mg/kg) i.p
Group III	Panchagavya Ghrita (4680 mg/kg ,p.o once a day for 15 days)
Group IV	Pancgagavya Ghrita (6240 mg/kg ,p.o once a day for 15 days)

Elevated Plus Maze Model^8,9,10

The elevated plus-maze apparatus consists of two open arms (16×5cm) and two closed arms (16×5×12cm) that extend from a common central platform. The entire maze is elevated to a height of 25cms above the floor level. Albino mice of either sex, weighing about 20-30g were divided into four groups of 6 animals each. Group I was served as control. Group II was treated with diazepam (2mg/kg i.p). Group III and IV were treated with two different doses of Panchagavya Ghrita (4680mg/kg,6240mg/kg) for 14 consecutive days. On the fifteenth day one hour after administration of the standard/test dose in respective groups, the mice were placed in a sound attenuated room, individually in the center of the maze facing one of the open arms. During a test session of five minutes, the parameters such as number of entries into the open arms, and time spent in the open arms were recorded.

Light-dark transition test^8,11,12

The light-dark apparatus consists of two-compartment chamber (40×60×20cm) comprising of a brightly illuminated area and a dark area separated by a wall with an opening at the bottom. Albino mice of either sex weighing about 20-30g were divided into four groups of 6 animals each. Group I was served as control. Group II was treated with diazepam (2mg/kg i.p). Group III, IV was treated with two different doses of Panchagavya Ghrita (4680mg/kg,6240mg/kg) for 14 consecutive days. On the fifteenth day one hour after administration of the standard/test dose in respective groups, mice were placed individually in the illuminated part of the cage and the parameters such as number of transitions between the light and dark area and time spent in the illuminated part of the cage were recorded during a test session of 5 minutes

Open field test^{13,14, 15}

The apparatus consists of a wooden box (40x40x50 cm). The floor of the box was divided into sixteen squares and the center of the field was marked to differentiate from other squares. The apparatus was illuminated with a 60-W lamp suspended 100 cm above. Albino mice of either sex weighing about 20-30g were divided into four groups of 6 animals each. Group I was served as control. Group II was treated with diazepam (2mg/kg i.p). Group III, IV was treated with two different doses of Panchagavya Ghrita (4680mg/kg,6240mg/kg) for 14 consecutive days. On the fifteenth day one hour after administration of the standard/test dose in respective groups, the mice were placed in the center of the arena and the parameters such as total number of squares crossed, total number of rearings, and time spent in the central square were observed and recorded during a test session of 5 minutes.

Statistical analysis:

All the data were expressed as Mean ± SEM and was analysed by using one-way analysis of variance (ANOVA), followed by Multiple comparison post hoc Scheffe’s test using SPSS software version 16. P value less than 0.05 was considered as statistically significant.

RESULT:

Elevated Plus Maze model:

The standard drug treated group (diazepam 2mg/kg) showed significant increase (p<0.05) in the number of open arm entries and time spent in open arms when compared to the control group. Panchagavya ghrita at a dose of 4680mg/kg and 6240 mg/kg, significantly (p<0.05) in a dose dependent manner increased the exploration activity in the open arm and also the time spent in the open arms in a similar way to that of the standard. The results are tabulated below

Table 1 Effect of Panchagavya ghrita on number of open arm entries and time spent in open arms in Elevated Plus maze

Sl. No.	Groups	Number of open arm entries	Time spent in open arms (sec)
1.	Control	13.6 ± 0.65 ^b	125.33 ± 0.84 ^b
2.	Diazepam /Standard (2mg/kg)	34.33 ± 1.44 ^a	178.67 ± 0.84 ^a
3.	Panchagavya ghrita (4680 mg/kg)	17.00 ± 1.23 ^b	133.33 ± 3.8 ^b
4.	Panchagavya ghrita (6240 mg/kg)	24.83 ± 1.79 ^{a, b}	140.67 ± 3.32 ^{a ,b}

Light/ Dark transition test:

The standard drug treated group (diazepam 2mg/kg) showed significant increase (p<0.05) in the number of entries into the light chamber and time spent in the light chamber when compared to the control group. Panchagavya ghrita at a dose of 4680mg/kg and 6240 mg/kg, significantly increased (p<0.05) the number of entries and time spent in the light chamber in a dose dependent manner similar to that of the standard. The results are shown in Table below

Table 2 Effect of Panchagavya ghrita on number of entries and time spent in the light chamber in Light/dark transition test

Sl. No	Groups	Number of entries into light chamber	Time spent in the light chamber (in sec)
1.	Control	13.33 ± 1.085 ^b	125.6 ± 0.71 ^b
2.	Diazepam / Standard (2mg/kg)	23.66 ± 1.115 ^a	170.50 ± 0.76 ^a
3.	Panchagavya ghrita (4680 mg/kg)	15.66 ± 1.054 ^b	130.50 ± 1.11 ^{a, b}
4.	Panchagavya ghrita (6240 mg/kg)	19.16 ± 1.075 ^a	146.67 ± 0.66 ^{a, b}

Open field test:

The standard drug treated group (diazepam 2mg/kg) showed significant increase (p<0.05) in the number of rearings, number of squares crossed and time spent in the central square when compared to the control group. Panchagavya ghrita at a dose of of 4680mg/kg and 6240 mg/kg, in a dose dependent manner significantly increased (p<0.05) the number of rearings, number of squares crossed and time spent in the central square in a similar way to that of the standard. The results are shown below

Table 3 Effect of Panchagavya ghrita on number of rearings, number of squares crossed and time spent in the central square in Open field test

Sl. No.	Groups	Number of rearings	Number of squares crossed	Time spent in the central square (sec)
1.	Control	17.166 ± 1.30 ^b	72.66 ± 1.52 ^b	31.166 ± 1.07 ^b
2.	Diazepam /Standard (2mg/kg)	43.83 ± 1.24 ^a	165.00 ± 1.82 ^a	84.66 ± 1.40 ^a
3.	Panchagavya ghrita (4680 mg/kg)	22.00 ± 1.06 ^b	82.66 ± 0.95 ^{a, b}	45.66 ± 1.22 ^{a, b}
4.	Panchagavya ghrita (6240 mg/kg)	30.50 ± 0.99 ^{a, b}	101.11 ± 1.42 ^{a, b}	67.16 ± 1.24 ^{a, b}

All the values are expressed as mean±SEM (n=6). a=p<0.05 when compared with Control b=p<0.05 when compared with Standard

DISCUSSION:

Anxiety is one of the highly existing nervous disorders which has highly affected about 1/8^th of the world population¹⁶. Currently, studies are focused on the development of appropriate treatment using natural resources^17,18. Panchagavya Ghrita has various medicinal properties which has been used traditionally to treat different ailments and by the recent study it is seen that it has been successfully used to treat epilepsy¹⁹ Panchagavya ghrita (PG) mainly consist of five elements such as cows urine, milk, curd, ghee and dung. All these comopents of PG show specific activity towards CNS. According to Ayurvedic formulary of India (IFA) Panchagavya ghrita is indicated in the treatment of Epilepsy (apasmara), fever (jawara), psychiatry diorders (unmada), Atattvabhinivesha (Obsessive compulsive disorder) and jaundice (kamala)²⁰ Despite the traditional use, no much scientific reports were available on the evaluation of the anxiolytic effects of the Panchagavya Ghrita. Cow milk maily contain omega -6 essential and omega -3 fatty linoleic acid which plays a significant part in inhibiting brain disorders. Cow urine is rich in volatile fatty acid which are very potent antioxidant agents²¹. Use of cow ghee help a person to keep normal function of physical entities and mental factors such as intellect and memory. From all above data it is clear that PG possesses anti- oxidant, neuroprotective activity and positive impact on memory power. In Ayurveda many of the psychiatric disorders where listed under unmada. The main advantage of ghrita is that they are capable of using as a single drug or in combination with other nootropic and mood stabilizing drug. Panchagavya ghrita prevents the etiology of unmada at the level of constitution, diet and life style. Anti-oxidant property of cow urine is able to repair degenerative changes in brain. The chemical changes in the brain get corrected by panchagavya ghrita^.

The current study was carried out to evaluate anti-anxiety activity of the Panchagavya Ghrita using animal models based on exploratory behaviour: Elevated plus maze (EPM), Light/ dark transition test, and Open field test. It was found that Panchagavya Ghrita possess significant anti- anxiety activity in a dose dependent manner. However there is a need to further elucidate the cellular and molecular mechanisms involved in the in the anti anxiety activity of panchagavya ghrita. Evaluvation of panchagavya ghrita for CNS related activities using few more models may further justify the therapeutic benefits in the treatment of anxiety disorder.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors are thankful to the Nitte (Deemed University) for providing necessary facilities to carryout this project.

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Received on 05.08.2021 Modified on 03.01.2022

Research J. Pharm. and Tech 2022; 15(11):4897-4900.

DOI: 10.52711/0974-360X.2022.00822