The project was executed in a planned manner. The steps that followed were:
- Field survey
- Scientific literature survey of identified plants
- Scientific evaluation (safety and efficacy) of selected medicinal plants
- Field Survey
The team of researchers initially worked to identify those areas where cultural folk medicines were strongly used. To carry out the survey, a friendly relationship was established with the households. For each meeting, target villages were selected where the objectives of surveys were discussed both volunteers and recommended individuals were identified as possible candidates to participate in personal interview for the survey.
The following activities were carried out.
- Women were interviewed in cultural sensitive manner by female members of the team.
- Herbalists within that village were identified.
- Each herbalist was interviewed by a member of the team. This interview was based on standard questionnaires and included enquiries of different types of plants collected from forest, farms, garden, and bought from the market.
- The data was recorded on the questionnaire and analysis was done.
For ethanobotanical assessments, following points were considered:
- Preparation before field work:
It included collection of ethnobotanical data related to women diseases from the literature, secondary information-regarding map, flora, land, the people, and the conservation issues in the region. Specific sites were selected with the help of maps before field visits.
- Formation of the multidisciplinary team:
Team for survey included gynecologist, taxonomist, pharmacist, microbiologist, chemist, biochemist, and anthropologist.
- Identification of fields:
All relevant information about the flora, soil, culture, and people was obtained before visiting the field.
- Ensuring community participation:
During survey, the villagers were informed about aims and objectives of the survey in a friendly atmosphere. A gynecologist was essentially included in the survey team so that people who usually do not have easy access to doctors could discuss their diseases and medications with the doctors.
Patients, old people, midwives and hakims were interviewed to record traditional knowledge of medicinal plants, used by them to treat women diseases. Interviews also included inquiries into types of plants collected from forests, farms, and gardens or brought for the market.
- Selection in choice of techniques:
Samples were collected from the patients by the microbiologist, which were examined in detail in various laboratories.
- Systematic work:
The survey was carried out systematically. It included the maps of the sites to be visited, complete data of the patients, geographical information of the village surveyed, identification of the biological samples collected from patients, and scientific evaluation of the ethnobotanical information. The conclusion of the survey was drawn with graphics and charts during the stay period.
Key Area for Questionnaire is:
- Name of the disease (in local language) along with symptoms.
- Local name of the plants.
- Types of plants.
- Parts of the plants used.
- Mode of preparation.
Availability and non-availability of plants used i.e. whether the plant had become more difficult to find in recent years.
- Optimal time for plant collection.
- Area of collection.
- Can a particular plant be substituted by another for same ailments?
- Personal experiences and treatment time.
- Any recurrences of the disease condition.
The data were recorded on the questionnaire and analysis was carried out:
Data Collection Included:
- Collection and identification of the plants and other material.
- Preparing ethnobotanic referee collection.
- Herbaria and curation of the plant and other species specimens.
- Judging the completeness of a plant survey.
- Collecting plants for photochemical analysis.
The main objective of the survey was to document and analyze information on the traditional use of medicinal plants by local population in rural areas of Sindh for the treatment of folkwomen diseases.
- Scientific literature survey of identified plants
A database of traditional medicines used for the treatment of women diseases has been compiled, which is a frame-based on-line reference of all information on medicinal plants and extracts used in the rural areas of Sindh. A monograph has been published.
This database and monograph include information about women diseases as well as plants, and herbs used in rural areas of Sindh for the treatment of these diseases. The data also include relevant herbal formulations and scientific evaluation.
- Scientific evaluation (safety and efficacy) of selected medicinal plants against skin diseases
All plants and other vegetations identified through the survey were collected or purchased, and extracted at H. E. J (International Center for Chemical and Biological Sciences). A systematic study was conducted on the chemistry and pharmacology of extracts and compounds isolated from natural sources. Extraction, isolation, and structure of active constituents were determined by using sophisticated spectroscopic techniques, and chemical methods.
- Extraction and Isolation
Plants were collected or purchased and dried in the air. They were crushed and soaked in methanol/ethanol for one week at 25o C. The crude extract was dissolved in distilled water, and defatted with hexane. The defatted aqueous extract was further fractionated with Chloroform (CHCl3), Ethyl acetate (EtOAc), and Butanol (BuOH). These extracts were evaporated, and evaluated for their antibacterial, antifungal, and other relevant assays. The active extracts were subjected to column chromatography (CC) on silica gel, sephadex LH-20, and HPLC, and eluted with gradients of different solvents like hexane-dichloromethane, hexane-ethyl acetate, dichloromethane-ethyl acetate, dichloromethane-methanol, water-methanol, etc., to yield the most active constituents from plants, and medicinal herbs. The structure elucidation of active constituents was carried out by using UV, IR, Mass, 1- and 2-D NMR techniques, and by chemical methods.
- Bioassay and Pharmacological Evaluation
Extracts (80% EtOH/MeOH-H2O) of identified plants were prepared and dried under vacuum. These extracts were screened for relevant activity (reputed therapeutic activity) by using high-throughput biological and pharmacological screening protocols at the Dr Panjwani Centre for Molecular Medicine and Drug Research (ICCBS). The bioassay screenings included:
Systematic Flow Chart of Survey Methodology and Plant Screening
In Vitro Studies for Treponema palladium (causative agent of syphilis)
- palladium was adjusted to 2×107 to 2.5×107 organism/mL. Different concentrations of extract/compounds were diluted and added to treponemes. After 24 h at 37oC in an environment of 5% CO2, motile organisms were assessed. Both live and dead organisms were counted.
In Vivo Studies for Treponema palladium
Adult female rabbits were injected intradermally with viable T. palladium. When (usually in 2 weeks) skin lesions were clinically apparent and animals were treated intramuscularly with different doses of the plant extract / pure compounds. As positive control, penicillin G at 23,000 U/kg was injected to a separate group of rabbits. As negative control, the rabbit was not treated and obscured.
In Vitro Studies for Candida albicans (causative agent of syphilis)
Minimal inhibitory concentration (MIC Testing) was assessed by broth micro dilution assay by using medium at pH 7, an inoculums size of 50 to 100 CFU/MI, and 24 h of incubation. Results of crude extract/pure compounds were compared with amphotericine B and Clotrimazole.
In Vivo Studies for Candida albicans
Mice were infected by intravenous administration of 5×106 CFU per animal from a freshly prepared 24-h C. albicans culture. An extract/pure compound was administered orally to mice (appropriate dose) over the period of 5-7 days.
In Vitro test for Neisseria gonorrhoeae (causative agents of gonorrhea)
The MICs and the extract/pure compounds were determined by agar dilution techniques. Several dilutions of the antimicrobial agents were distributed in to Mueller-Hiniton agar. The culture was then inoculated on to the antimicrobial agents, containing plates. The plates were incubated for 18-24 h at 35oC in a CO2 atmosphere.
In Vitro test against Chlamydia trachomatis
Lodo-oxyyuridine treated McCoy cells were infected with an inoculum of C. trachmatis. A serial of several fold dilution of extracts/pure compounds was added after 48 h inoculation of pathogens and incubation continued for a further 48 h and then the MIC was noted.
In Vivo Studies for Female Sexual Dysfunction
Female rabbits were selected as animal model to examine female genital arousal response. The changes in vaginal and clitoral hemodynamic in response to pelvic nerve stimulation were recorded.
In Vivo Studies for Premature Menopause
Primodial germ cells deficient mice were used as animal model for the pathogenesis and treatment of premature menopause.
Crude extract/pure compound used in women disease were subjected to cytotoxicity assay. Crude extracts/pure compounds were subjected to cytotoxicity assay by using human neutrophils. The assay was based on the reduction of tetrazolium salt WST-1 by mitochondrial dehydrogenases of viable cells to yellow organ formation dye, which was measured spectrophotometrically.