Methodology example in research

CHAPTER THREE

MATERIAL AND METHODS

3.0 Introduction

This section presents the study in line with the materials and methods to be used in line with study objectives.

3.1 Ethnopharmacological survey of medicinal of plants with anti-ulcer activitiy in Uganda

Methods

An ethno pharmacological survey (questionnaire-based cross-sectional descriptive study) will be used.

3.1.2 Study Area

This will include selected districts of Uganda were herbal medicines is widely practiced.

3.1.3 Selection of informants

An ethno pharmacological survey (questionnaire-based cross-sectional descriptive study) will be used. The study will be conducted in accordance with National council of science and technology guidelines. The objectives of the study will be explained to the participants and a written informed consent will be obtained from the participants, Atotal of 150 herbalists will be interviewed in this study. All the informants will be asked to provide information on the plants that they use to treat gastrointestinal diseases, parts of the plants used such as leaves, roots, flowers, stems and seeds, methods of preparations like (decoction, juice, infusion, powders) and methods of administration, either orally or topically. In addition to their opinion about advantages of herbal medicine. This method has proven to be very practical and useful in data collection, duration of the interviews will range from 20 to 60 minutes. Interviews shall be conducted in local languages of the informants and the plant names are to be given in local languages and later translated in to English and latin using reference books.

3.1.4 Plant identification and data analysis

The collected plant samples from these informers are to be stored in the laboratory in the chemistry laboratory Kyambogo University in appropriate glass ware and wooden frames and later to be authenticated by a specialist.

3.1.5 Data analysis

The frequency of citation (FC) for all plants species in this study were calculated by using the following formula:

FC = (Number of times a particular species was mentioned by herbalists and traditional healers/a total number of occasions that all species were mentioned) × 100 [24].

To evaluate the relative importance of plants in indigenous healthcare systems, the use value (UV) is used as a micro-statistical tool, which reflects people interaction with specific plants as the best treatments for ulcers. It is a quantitative method that can be used to prove the relative importance of species known locally. It can be calculated according to the following

equation: UV ¼ PU n

Where UV is the use value of a species; U is the number of citations per species; n is the number of informants Factor of informant’s consensus (Fic) will be calculated according to the following equation:

Fic ¼ Nur−Nt Nur−1

Where Nur is the number of use citations in treatment of an intestinal disease category, and Nt is the number of taxa used for the treatment of these diseases.

3.2 Phytochemical screening and investigation of selected medicinal plant extracts.

Collection of plant material

The whole plant or its parts will be collected from the known sources. the collected plant specimens will be identified and authenticated from the official sources.

3.2.1 Processing of plant material

Collected plant material will be subjected to processing such as drying, grinding, sieving to make it suitable for extraction.

3.2.2 Extraction of plant material with various solvents  

Coarse powder form of plant material will be subjected to successive extraction. The solvent and extraction method will be optimized according to nature of Phyto-chemical.

3.2.3 Phyto-chemical testing

All the extracts will be subjected to Phyto-chemical testable screening used in standard methods.

Standardization of extracts will be carried for estimation of alkaloids, saponins , tannins polyphenols, and flavonoids.

3.4 Pharmacological evaluation of plant extracts for anti-ulcer activity (in vitro and in vivo methods).

After preliminary phytochemical evaluation of plant extracts/ fraction, the most effective plant extract will be subjected to suitable invitro and and or invivo preclinical models.

3.4.1 Bio-activity guided fractionation and identification of anti-ulcer compounds in the plant extracts.

The most bioactive potent plant extract from pharmacological screening will be subjected to bio-activity guided fractional separation and isolation, wish will be carried out by using pertinent separation techniques, such HPLC, TLC

3.5 Structure elucidation of the bio-active compound in the plant extracts.

The separated, isolated and purified phytochemical from the Bioactive extracts/fractions will be characterised using suitable spectroscopic techniques such as NMR, IR, MS, UV.

PROPOSED WORK PLAN

1. Collection, identification and authentication of selected medicinal plant

material.

2. Processing of crude drug for extraction
3. Extraction of plant material:
4. a) Selection of solvent for extraction
5. b) Selection and optimization of extraction method
6. Phytochemical screening and standardization of plants extracts:

Phytochemical screening of both qualitative and quantitative analysis for TLC

fingerprinting of extracts and standardization of extracts by estimating total

carbohydrates, proteins, lipids, polyphenols, flavonoid, saponin, alkaloids,

steroids content etc

5. Pharmacological screening of plant extract(s)/fraction(s) for:
6. i) Acute oral & repeated dose toxicity
7. ii) Antioxidant activity

iii) Anti-inflammatory activity

1. iv) Anti-ulcer activity
2. Bioactivity guided isolation of Phyto-constituents from fractions or extracts:

The most bioactive and potent extract(s)/ fraction(s) resulted from afore

mentioned screening will be subjected to bioactivity guided fractionation,

separation and isolation, which will be carried out by using pertinent

separation techniques.

7. Characterization and pharmacological screening of bioactive phytoconstituent/s:

Separated, isolated & purified phytoconstituent/s from bioactive extract(s)/

fraction(s) will be characterized using suitable spectroscopic techniques. The

isolated phytoconstituent/s will be screened by selected pharmacological

evaluation methods