Pharmacognostical, Phytochemical and Pharmacological studies on the roots and rhizomes of Nymphoides hydrophylla (Lour.)Kuntze




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Pharmacognostical, Phytochemical and

Pharmacological studies on the roots and

rhizomes of Nymphoides hydrophylla (Lour.)Kuntze



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BRIEF RESUME OF THE INTENDED WORK




NEED FOR STUDY

Herbal medicine is the oldest form of health care known to mankind. The plant kingdom is a virtual goldmine of potential drug targets and other active constituents awaiting to be discovered¹. In India, the Vedas are the epic poems, which contain rich material on the herbal medicines of that time (Rig Veda)2. In the works that followed the Vedas, “Ayurveda” in particular gives in detail the properties of various drugs³. “Charaka Samhita” gives description and details of around 350 herbal medicines most of which are still continued to be gathered from wild source to meet the demand of the health care profession4. Despite the rich heritage of knowledge on the use of plant drugs, little attention had been paid till the latter part of 19th century5. A recent surgence of interest in alternative health care and natural products medicine, the regulatory and manufacturing developments has resulted in world wide use and increased market demand for herbal medicines around the globe6.


Even though nearly 3,00,000 species of higher plants are available, a very small portion has been investigated for medicinal properties7. Hence there is a need to investigate the plants for their therapeutic uses following scientific methods.
Herbs are the attractive alternative medications to many patients with sleep disorders, who may be averse to use conventional drugs8. Many drugs such as Valeriana jatamansi, Cannabis sativa, Withania somnifera have been traditionally used as sedative9, but more research is needed on new plants with sedative activity and on its use in sleep disorders and in calming the patient.
The term ‘Epilepsy’ is collectively designated for a group of chronic CNS disorders characterized by spontaneous occurrence of seizures generally associated with loss of consciousness and body movements (convulsions). Modern drug therapy is complicated having many side effects, teratogenic effects and long term toxicity. Medicinal plants used for the therapy of epilepsy in the traditional system of medicine have shown to possess promising anticonvulsant activity in animal models. Therefore anticonvulsant screening can be an invaluable source for identifying new anti-epileptic drugs10.
The plant selected for the study is Nymphoides hydrophylla (Lour.) Kuntze (= Nymphoides cristatum (Roxb.) O. Kuntze). Nymphoides Hill is a genus of aquatic flowering plants in the family Menyanthaceae. It consists of 20 species, out of which N. hydrophylla (Lour.) Kuntze, N. macrospermum Vasudevan, N. peltatum, N. indica (Lour.) Kuntze, N. sivaranjanii K.T. Joseph and N. krishnakesara K.T.Joseph and Sivar are found in India11. These aquatic herbs have submerged roots and floating leaves that hold the small white or yellow flowers above the water surface12, 13, 14.
According to ayurvedic literature the accepted source for the drug ‘Tagara’ is Valeriana jatamansi Jones. In South India, drug by name Granthika Tagara (Kannada) belonging to N. macrospermum is used as one of the sources in place of Tagara in several therapeutic preparation for the treatment of various diseases such as anaemia, epilepsy, fever, jaundice, mental disorders and also as brain tonic15,16,17,18.
As Nymphoides hydrophylla is an allied species of N. macrospermum it is likely to possess similar activity. Hence the present study is aimed to carry out the sedative and antiepileptic activity on the roots and rhizomes of Nymphoides hydrophylla 19, 20,21,22.


REVIEW OF LITERATURE

  • Isolation of a triterpenoid, Bet-20(29)-en-3-ol-28-oic acid (1) from the ethyl acetate extract of Nymphoides cristatum (Roxb.) O.Kuntze and elucidation of its structure based on the spectral evidences. The compound was also screened for antimicrobial and cytotoxic activity. The compound was found to exhibit significant antimicrobial activity and prominent cytotoxic activity23.

  • Invitro study on the antimicrobial effect of extracts of coastal plants of Primorye among which Alisma orientale, Monochoria korsakowii, Euryale ferox and Nymphoides peltata showed highest inhibitory effect against dysentery pathogen24.

  • Screening of some medicinal plants like Lannea grandis, Adina cordifolia, Boswellia serrata, Limnophila racemosa, Limnophila indica, Exacum pedunculatum, Waltheria indica and Nymphoides cristatum for antibacterial activity was carried out. Among them Waltheria indica and Nymphoides cristatum showed less antibacterial activity25.

  • Invitro plant regeneration in Nymphoides cristatum (Roxb.) O. Kuntze26.

  • Study on the cell wall ultrastructure of the testa of Nymphoides hydrophylla (Roxb.) O. Kuntze27.


AIMS AND OBJECTIVES OF THE STUDY

AIMS


1. To undertake Pharmacognostical investigation on roots and rhizomes of Nymphoides hydrophylla.

2. To investigate and evolve phytochemical parameters for drug.


3. To investigate and evaluate the sedative and anti-epileptic activity of the drug.


OBJECTIVES
1. To study the taxonomical characters of the plant which help in the

investigation.


2. To understand the macro and microscopical characters of the drug besides phytochemical analysis, which helps in evolving diagnostic characters for the identification of the drug and also contribute towards pharmacopoeial standards.

3. To carry out toxicological studies.




  1. To evaluate the sedative and anti-epileptic activity of the drug.


MATERIALS AND METHODS

SOURCE OF DATA
1. Literature survey, Helinet, Medline, Pubmed, Science direct, Google,

Chemical Abstracts.


2. Journals and other Publications
3. Lab based studies.
METHOD OF COLLECTION OF DATA

Field and laboratory based studies.
Field Work:

The selected plant’s roots and rhizomes will be collected from wild source. A voucher specimen will be collected and deposited at the herbarium/museum of the department.



Laboratory work:



  1. Taxonomical studies:

The plant material will be identified by using various Floras like Flora of Hassan district28 and Flora of the Presidency of Madras29. Herbarium specimens will be prepared following the method of Jain and Rao as per International standards30.


B. Pharmacognostical studies:

Free hand sections of the drug will be taken following Wallis31. Microscopical investigation and histochemical tests will be carried out following Evans32.


Quantitative microscopy for carrying out the measurement of tissues will be done by using stage and ocular micrometers. Photomicrographs will be taken and the images captured on the computer.

C. Phytochemical studies:
Physical constants of the drug will be determined as per Indian Pharmacopoeia33, WHO guidelines34and phytochemical tests for detection of organic constituents will be done as per Harborne35. The chromatographic studies following Krebs et al36, HPTLC studies following Wagner et al37 and fluorescence studies following Chase and Pratt38 will be carried out.
D. Pharmacological studies:
ANIMALS:-

Swiss albino mice of either sex in the weight range of 18-25 g will be used for acute toxicity studies. Animals outside this weight range will not be included in the study.
ACUTE TOXICITY STUDIES:-

The acute toxicity tests aims at establishing the minimum lethal dose of the drug extracts.They will be carried out following the method described by Ghosh 2005 39. The extracts will be orally administered to seperate groups of Swiss albino mice at different dose levels and the animals will be observed for symptoms of toxicity/death.

Total number of mice required-50
SEDATIVE ACTIVITY:-

Alcohol and aqueous extract of the drug will be evaluated for sedative activity using the following models.




  1. Potentiation of pentobarbitone-induced sleeping time 40,41

Sedative, in general, is a drug that reduces excitement and calms the person. Hypnotics, sedative and tranquilizers are known to prolong sleep time after single dose of pentobarbitone.

The criteria for the measurement are induction of sleep indicated by loss of righting reflex and duration of sleep. Mice are used since the metabolic elimination of pentobarbitone is rapid in this species.
Each group comprises of 6 mice.


  • Group-1:- Control group -Pentobarbital sodium (45mg/kg body weight, i.p.)

  • Goup-2:- Standard group -Diazepam (3mg/kg body weight, i.p.)+ pentobarbital sodium (45mg/kg body weight, i.p.).

  • Group 3 and 4:- Test groups- 2 doses each of aqueous extract (p.o.) + pentobarbital sodium (45mg/kg body weight, i.p.).

  • Group 5 and 6- Test groups-2 doses each of alcohol extract (p.o.) + pentobarbital sodium (45mg/kg body weight, i.p.).




  1. Locomotor activity using actophotometer 40,41

Most of the drugs acting on central nervous system influence the locomotor activities in man and animals. The locomotor activity (horizontal activity) will be measured using an actophotometer which operates on photoelectric cells which are connected in circuit with a counter. When the beam of light falling on the photocell is cut off by the animal, a count is recorded which is directly proportional to its locomotor activity.

Each group comprises of 6 mice.


  • Group-1:- Control group-Vehicle.

  • Group-2:- Standard group- Diazepam (4mg/kg body weight, i.p.).

  • Group 3 and 4:- Test groups- 2 doses each of aqueous extract (p.o.) + Diazepam (4mg/kg body weight, i.p.).

  • Group 5 and 6:- Test groups-2 doses each of alcohol extract (p.o.) + Diazepam (4mg/kg body weight, i.p.).

Total number of mice required-36+36=72.


ANTI-EPILEPIC ACTIVITY:-
Anti-epileptic activity of the alcohol and aqueous extract of the drug will be evaluated using the following models.


  1. Maximal electroshock seizures (MES) method 40

The maximal electro-shock (MES)-induced convulsions in animals resemble grandmal type of epilepsy in man. In this method electric shock will be applied through the corneal electrodes. Through optic stimulation cortical excitation is produced. The MES-convulsions are divided into five phases such as (a) tonic flexion, (b) tonic extensor, (c) clonic convulsions, (d) stupor, (e) recovery or death. A substance is known to possess anticonvulsant activity if it reduces or abolishes the extensor phase of MES induced convulsions.


Each group comprises of 6 mice.


  • Group-1:- Control-Electric shock (150mA current for 0.2 sec)

  • Goup-2:- Standard group- phenytoin (2.5mg/kg body weight, i.p)+ Electric shock (150mA current for 0.2 sec)

  • Group 3 and 4:- Test groups-2 doses each of aqueous extract (p.o.)+ Electric shock (150mA current for 0.2 sec)

  • Group 5 and 6- Test groups -2 doses each of alcohol extract (p.o.)+ Electric shock (150mA current for 0.2 sec)



  1. PTZ – induced convulsions 40,41

Chemo-convulsions due to pentylenetetrazol produce clonic-type of convulsions resembling petitmal type of convulsions in man. PTZ is a central nervous stimulant and it produces jerky type of clonic convulsions in mice and rats.


Each group comprises of 6 mice.


  • Group-1:- Control- PTZ (70mg/kg body weight, i.p.).

  • Group-2:- Standard group- Diazepam (4mg/kg body weight, i.p.)+ PTZ (70mg/kg body weight, i.p.).

  • Group 3 and 4:- Test groups- 2 doses each of aqueous extract (orally) + PTZ (70mg/kg body weight, i.p.).

  • Group 5 and 6:- Test groups- 2 doses each of alcohol extract (orally) + PTZ (70mg/kg body weight, i.p.).

Total number of mice required for anticonvulsant activity studies-36+36=72.

The total number of mice required for pharmacological activities -194.
Results of the studies will be subjected to statistical analysis by One-way ANOVA followed by Dunnett’s multiple comparison tests.
Does the study require any investigation to be conducted on patients or other humans or animals? If so, please describe briefly.
No
Has ethical clearance been obtained from your institution in case of

2.3?

Yes.
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