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ORIGINAL ARTICLE
Year : 2017  |  Volume : 37  |  Issue : 1  |  Page : 31-36

Experimental evaluation of Hygrophila Schulli seed extracts for antistress activity


1 Department of Pharmacognosy, SCES's, Indira College of Pharmacy, Tathwade, Pune, Maharashtra, India
2 Department of Pharmacognosy, KLE University's College of Pharmacy, Belgaum, Karnataka, India
3 Department of Pharmacognosy, JSPM'S, Rajarshri Shahu College of Pharmacy, Tathwade, Pune, Maharashtra, India

Date of Web Publication13-Jul-2018

Correspondence Address:
Dayanand Kannur
Department of Pharmacognosy, SCES's, Indira College of Pharmacy, Tahthawade, Pune, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/asl.ASL_191_17

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  Abstract 

Background: Stress is the causative factor for various diseases and disorders faced by majority of the diseased population. The seeds of Kokilākṣa Hygrophila schulli are attributed with Rasāyana properties as per the Ayurvedic literature. Considering the above, the seed extracts of H. schulli were screened for antistress activity in animals. Aim: To investigate the adaptogenic activity of ethanolic and hexane extracts of Hygrophila schulli seeds using in vivo models. Materials and Methods: The ethanolic and hexane extracts of Hygrophila schulli were subjected to qualitative chemical analysis to detect the presence of various phytoconstituents. The extracts were subjected HPLC analysis. The chromatographic analysis was carried out which revealed the multicomponent and complex nature of the extracts. The seed extracts of H. schulli were screened for antistress activity using Swim Endurance test in mice and Cold-Immobilization Stress model in rats to ascertain the Adaptogenic potential. Results: HPLC analysis confirmed the presence of flavonoid Quercetin, the ethanolic and hexane extracts were found to increase the swim endurance time, both extracts lowered the elevated blood glucose, cholesterol as well as triglyceride levels in cold immobilization stress model and maintained normal homeostasis. Conclusion: Hygrophila schulli enhances the physical endurance as well as normalizes the body imbalance due to stressors. The seeds of Hygrophila schulli thus possess adaptogenic property.

Keywords: Adaptogenic, antistress, cold immobilization Hygrophila schulli, swim endurance, stress


How to cite this article:
Kannur D, Nandanwadkar S, Dhawane S, Phulambrikar S, Khandelwal K. Experimental evaluation of Hygrophila Schulli seed extracts for antistress activity. Ancient Sci Life 2017;37:31-6

How to cite this URL:
Kannur D, Nandanwadkar S, Dhawane S, Phulambrikar S, Khandelwal K. Experimental evaluation of Hygrophila Schulli seed extracts for antistress activity. Ancient Sci Life [serial online] 2017 [cited 2018 Dec 16];37:31-6. Available from: http://www.ancientscienceoflife.org/text.asp?2017/37/1/31/236546


  Introduction Top


Every individual on this earth experiences stress. Extreme Stress is harmful for the body and hence needs to be treated. Stress leads to the pathogenesis of various diseases and psychiatric disorders such as cognitive dysfunction, psychological depression, anxiety, immune suppression, diabetes, impotence, gastric ulcers, ulcerative colitis, etc.[1]

Adaptogens are naturally occurring antistress natural compounds proven to act against and counter stressful conditions.[2] These substances tend to increase the nonspecific resistance of the individual to various stressors which threaten and disturb the normal internal homeostasis. Adaptogens increase tolerance to change in environmental conditions and resistance to noxious stimuli such as exposure to cold, heat, pain, general stress, infectious organisms.[3] These agents are basically preventive rather than curative in action and have been claimed to arrest ageing process.

Hygrophila schulli

Hygrophila, commonly known as the temple plants or hygros, are flowering wild plants belonging to the family Acanthaceae [Figure 1]. They are known as Tālmakhānā, Ikṣura, Ikṣugandha, and Kokilākṣa [Figure 2]. Its seeds are of a shape similar the eyes like the Indian Cuckoo.[4] The plant is commonly found in moist places on the banks of rivers, ditches and paddy fields throughout India.
Figure 1: Hygrophila schulli plan

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Figure 2: Seeds of Hygrophila schulli

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Ayurvedic literature has references to the use of seeds and roots of Kokilākou. The bitter seeds are known to be aphrodisiac. It is a general tonic and in pregnant women it is used as a sedative. In the treatment of spermatorrhoea and impotency, the seeds are administered with milk and honey. In diarrhoea the seeds paste is administered along with buttermilk.

Kokilāksh is said to be śītavīrya, madhuravipāka and used for the treatment of prameha (~diabetes), atisāra (dysentery).[5],[6] The complete plant, and its ash have found beneficial use in alternative medicine. Various diseases and disorders such as arthritis, inflammation and pain, jaundice, UT infections have been treated using the plant.[7],[8],[9] The plant is known to be aphrodisiac and general tonic due to its healing properties.[10],[11]

Ayurvedic Properties

Guṇa (Quality) Guru, Snigdha, Picchila

Rasa (Taste) Madhura

Vipāka (Metabolism) Madhura

Vīrya (Potency) Śīta

Prabhāva (Impact) Śukrajanana

The use and applications of Kokilākṣa have been mentioned in detail and a few are given below [12]



The properties attributed to Hygrophila are śukraśodhana (purifies/cleanses semen),[12],[15]santarpaṇa (provides nourishment),[12],[15]balya (tonic, strength promoter),[12],[15]vṛṣya (Aphrodisiac),[12],[14]kāmalāhara (alleviates jaundice),[12],[13]stanyajanana (galactagogue), āmahara (Eradicates intermediate products of digestion/metabolites),[12],[14]Śothaghna (Reduces swelling and inflammation).[12],[14]

The traditional practitioners have been using the Kokilākṣa or Tālmakhānā in various formulations, administered to patients for treating various diseases and disorders.

Vaidyaratnam Kokilākṣakam Kaṣāyam – is an Ayurvedic medicine used in treating jaundice, anemia and hepatic conditions. This medicine is based on Kerala Ayurveda principles. Kokilākṣa Kaṣāyam is administered with an intent to increase elimination of uric acid and other toxins from the body. Secondly, it has anti-inflammatory and detoxifying actions, which helps to reduce inflammation and detoxify the body. The combination of Tālmakhānā, Giloy, and Pippalī cūrṇa reduces āma formation in the body, improves digestion, modulates metabolism, and increases the elimination of āma toxins from the body. It is also available in Kaṣāyam tablet form which is effective in reducing uric acid level, Gout, and Rheumatoid arthritis.

Various ethnobotanical and traditional uses as well as phytochemical and pharmacological reports about Hygrophila auriculata (K. Schum) Heine were reported.[16] Significant hepatoprotective activity of the methanolic extract of the seeds in paracetamol induced hepatotoxicity has been reported.[17] The root extracts exhibited significant hepatoprotective action in CCl4-induced hepatotoxicity in rats and antioxidant activities were also reported.[18]Astercantha longifolia is a vājīkarana rasāyana herb and its effect on the sexual behaviour and reproductive parameters have been reported.[19]

The plant contains Sterols,[20],[21] Terpenoids, Alkaloids, and Flavonoids.[22] Its seeds and roots are a known remedy for arthritis. The seeds are reported to possess anti-inflammatory and antinociceptive activity.[23] The aerial parts have been shown to possess the Erythropoietic and Hepatoprotective activity.[17]

Kokilāksh is commonly used in various marketed formulations and is the main ingredient in products such as Confido, Lukol, Speman, Tentex Royal, Speman Vet,[24] Vitomanhills, Kokilākshkam Kashaya, Kokilākshkam Kvātha, Kokilāksh – G tablets.


  Materials and Methods Top


Collection and authentication

Plant material was collected from the local market, and was authenticated from Botanical survey of India, Pune. The specimens were preserved in the repository. Hygrophila schulli (Acanthaceae) (V. No. Kannur 1).

Extraction

The plant material was size reduced and was further extracted by maceration with 95% Ethanol HSE and Hexane HSH separately and further concentrated using Rota evaporator and dried under vacuum.

Phytochemical screening

The Hexane and Ethanolic extracts of Hygrophila schulli were subjected to qualitative analysis for the various Phytoconstituents such as Alkaloids, Carbohydrates, Glycosides, Phytosterols, Saponins, Tannins, Proteins, Amino acids and Flavonoids [Table 1] and [Table 2].
Table 1: Percentage yield of the extracts

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Table 2: Phytochemical evaluation of Hygrophila schulli

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High performance liquid chromatography studies

The extracts (5 mg) were dissolved in of the HPLC grade methanol (5 ml). A part this solution (10 μ L) was analyzed by high performance liquid chromatography (HPLC) using a Zorbax ODS C18 4.6 mm × 250 mm, 5 μm reverse phase column attached to Agilent 1100 high performance pump and Agilent 1100 variable wavelength UV detector (254 nm) using optimized solvent systems at a flow rate of 1 ml/min.

Pharmacological evaluation

The adaptogenic activity was screened using Swim Endurance Model, Cold Stress Model, Cold Stress with Immobilization Model.

Animals

Healthy Albino Wistar rats of either sex weighing between 150-200 gm and Swiss albino mice weighing 30-40 gm of either sex were used. The experimental animals were maintained under 12:12 h light dark cycle, in an animal house with controlled temperature. The animals had free access to water and Standard pellet diet. The Institutional Animal Ethics Committee approved the experimental protocol.

Adaptogenic activity

Swim endurance model

Hygrophila schulli ethanolic and hexane extracts were evaluated using this model at two different doses. Swiss albino mice were divided into five groups of six animals each in two sets and were treated with respective extracts and a standard drug for 9 days. On the 10th day the test was performed and the drowning time of the mice was noted and Swim endurance time was calculated.[17]

Immobilization and cold stress model

Hygrophila schulli ethanolic and hexane extracts were evaluated using this model at different doses. The Wistar albino rats were divided into seven groups of six animals each and were treated with respective extracts at a dose of 250 and 500 mg/kg b.w orally one hour prior to stress exposure daily for 10 days by immobilization [24],[25] in cold conditions. The animals were taken from their home cages and individually placed in polymer containers with partition to separate individual animals. The animals were immobilized by tying forelimbs and hind limbs using a woolen thread, the containers were placed inside a refrigerator such that the temperature to which the animals were exposed was 4°C,[25],[26] they were returned to their home cages after 4 h. This procedure was repeated for 10 days at a specific time period between 12:00 noon to 4:00 p.m. Geriforte (a multiherbal formulation of Himalaya Drug company) (43mg/kg b.w.)[27] was used as a standard drug. The animals were treated and were subjected to stress for 10 days and were free to have food and water, on the 10th day, one hour after exposure to stress the animals were sacrificed by decapitation, blood was collected from the arterial jugular and serum was separated.[28],[29] The serum was used for the estimation of various biochemical parameters using different biochemical kits (Biolabs/Span Kits).


  Results Top


High performance liquid chromatography analysis

The HPLC analysis of H. schulli ethanolic extract was carried out using Acetonitrile and water in the ratio 30:70, which exhibited six prominent peaks [Figure 3] which were polar in nature. The hexane extract was analyzed using medium polar system Acetonitrile and water in the ratio 70:30 which exhibited 11 prominent peaks [Figure 4] showing more complex nature of extract. The chromatogram further indicated that it was a mixture of nonpolar and medium polar compounds.
Figure 3: High performance liquid chromatography Chromatogram of Hygrophila schulli ethanolic extract

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Figure 4:Chromatogram of Hygrophila schulli hexane extract

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The presence of Quercetin in both the extracts was shown by taking HPLC chromatogram of Quercetin with both extracts in their respective solvent systems.

The Standard Quercetin is observed at Rt. 1.519 min in the H. schulli ethanolic extract [Figure 5]. The Standard Quercetin is observed at Rt. 3.515 in the H. Schulli Hexane extract [Figure 6].
Figure 5: High performance liquid chromatography chromatogram of Hygrophila schulli ethanolic extract with along Std. Quercetin

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Figure 6: High performance liquid chromatography Chromatogram of Hygrophila schulli hexane extract along with Std. Quercetin

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Adaptogenic activity

Swim endurance time in H. schulli seed extracts

In the case of Hygrophila schulli seed extracts the swimming time was observed to be significantly increased in the extract treated groups in comparison to the vehicle treated animals at both the doses of 250 mg/kg bw and 500 mg/kg bw. The hexane extract exhibited the most significant activity at both the temperatures which was higher than the standard drug [Table 3] and [Figure 7]. The ethanolic as well as the Hexane extracts at higher dose of 500 mg/kg bw were highly significant in increasing the swimming time in mice as well as the physical endurance. Even in case of 10°C temperature the animals were able to sustain the severe cold for a longer period.
Table 3: Swim endurance time in Hygrophila schulli seed extracts in minutes

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Figure 7: Swim endurance time in Hygrophila schulli extracts

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In case of Hygrophila schulli seed extracts, the ethanolic and hexane extracts exhibited moderate adaptogenic activity at 250 mg/kg bw. The Ethanolic and the Hexane extracts at a dose of 500 mg/kg bw., lowered the Blood sugar level, Cholesterol, Triglycerides as well as the LDL levels significantly (P < 0.0001) [Table 4] and [Figure 8].
Table 4: Evaluation of Hygrophila schulli seed extracts in cold and immobilization stress model

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Figure 8: Biochemical parameters in cold immobilization stress

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The ethanolic and hexane extract was found to be highly significant in lowering the elevated lipid levels in comparison to the standard drug too. It was also efficient in increasing the HDL levels. The Ethanolic as well as the Hexane extracts exhibited preventive action against stress induced alterations in the TLC and DC [Figure 9] and [Table 5]. The Ethanolic extract was significant in comparison to the Hexane extract in controlling the differential count, thereby maintaining the DC level near to the normal.
Figure 9:Total Leukocyte Count in Hygrophila schulli extracts treated animals

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Table 5: Total leukocyte count and differential count of Hygrophila schulli seed extracts in cold and immobilization stress model

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  Discussion Top


The results clearly exhibit that exposure to cold and immobilization leads to stressful conditions; the normal homeostasis is also disturbed leading to severe imbalance in the biochemical parameters. The severity of stress increases with immobilization along with cold exposure which further leads to clinically pathogenic conditions as observed in the disease control group. Exposure to stress leads to Hyperlipidemia as well as hyperglycemia, which have been reported earlier.[26],[27] The Hygrophila schulli seeds have been used as general tonic in Ayurveda, They have been used as aphrodisiac too indicating the ability to act as rejuvenators.[4],[30] These claims are substantiated in the present study.

The phytochemical and HPLC analysis indicate the seed to be a complex multi component entity. Flavonoids, sterols and Terpenoids were found to be present in the seeds. The presence of Quercetin is also confirmed.

The results evidently indicate the functional ability of the extracts in maintaining the normal homeostasis and controlling the alterations in the biochemical parameters caused by stress induction.[28],[29],[30],[31] It was observed that both the ethanolic as well as hexane extracts were more effective at higher doses. The Hyperlipidemia induced due to stress was significantly lowered. Both the extracts were able to lower the elevated blood glucose level and at the same time lower the elevated levels of Leukocyte count. This can be attributed to the presence of Terpenoids and Sterols in the extracts.[21]


  Conclusion Top


These results are encouraging enough to ensure the designing of adaptogenic formulations of H. schulli. The current working culture, sedentary lifestyle, pollution, stress levels and working culture are main contributory factors for various diseases and disorders, the results obtained here will help in developing formulations with antistress properties and also properties boosting the vigor and strength. The results obtained herein validate the Ayurvedic as well as folklore claims; the results support the antistress activity of Hygrophila schulli. The balya, santarpaṇa, vṛṣya, āmahara, śothaghna properties are exhibited by the drug.

Thus it can be concluded that the Hygrophila schulli seeds possess antistress properties and have the capacity to enhance the physical endurance, hence the Hygrophila schulli can be used as an adaptogenic agent.

Financial support and sponsorship

The first author is thankful to Director BCUD, Savitribai Phule Pune University for the financial assistance supporting the research work.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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