Gemotherapy, another way of phytotherapy

Acknowledgements:

I thank Ph.D. Fernando Pitera for recommending me to Ph.D. Antonio Graverini from Officine Terapie Innovative.

I thank Ph.D. Antonio Graverini for accepting my internship under his guidance.

Lots of thanks to the research, development and quality control laboratory collective, whom helped me and guided me in all my activities within this department.

I thank Ph.D. Iosif Ianculov for his friendship, his help and guidance in all my activities.

I thank Ph.D. Ioan Hutu for advising me and helping out through this entire period.

I thank my husband and family who supported me and encouraged me constantly

Thanks to everyone!

Chapter I. Bibliography

About the laboratory

Officine Terapie Innovative

S.S. Tiburtina Valeria Km 69,300-67061 Carsoli (AQ), Italia

Situated in Abruzzo one of the most pitoresque regions of Italy, OTI sits at the base of italian Homeopathy history.

The anterior experience from the pharmaceutical sector has made posible the creation of the first homeopathic laboratory in Italy.

The research, development and quality control laboratory is equiped with the following apparatus:

Spectrophotometry U.V. - visible

Thin Layer Chromatography (TLC)

Gas chromatography/mass spectroscopy GC/MS

Hight performance liquid chromatography (HPLC)

Microbiological anlysis

Quality control in all the phases of production

Atomical Absorbtion

TOC (Total Organic Carbon)

Steriltest millipore

Abstract

During this period i have documented myself from the bibliography of the O.T.I. library for my Ph.D. theme. I have prepared the gemoderivates from mushrooms (Agaricus bisporus, Pleurotus ostreatus, Lenitnula edodes) using the following recipee from the french pharmacopoeia: the embryonic tissues are left to macerate for 21 days in a alchool and glicerine mixture(mass report, dehidrated vegetal material: alchool and glicerine mixture 1:20). I 've made and learned different fizico-chemical analysis methods on all the existent apparatus in the laboratory. I have cutivated Crocus sativus (saffron) in vitro for obtaining explants(stamens or seedlings).

Gemotherapy, another way of phytotherapy

The founding father of modern gemotherapy is the belgian doctor Pol Henry, he redescovered the benefactor quality of the buds and he explained the existing paralelism between the evolution of the forest and the evolution of the human proteic matter. Pol Henry named this therapeutical method using different terms: gemotherapy, blastotherapy and phytoembriotherapy. This new discipline was detailed and developed further by the french homoeopath Max Tetau.

This is a therapeutical method of biotherapy (and a branch of phytotherapy) whitch uses first decimal dilluted solutions of hidroglyceroalchoolic macerates from fresh vegetal extracts, represented through meristematic tissues: gemmas, young branches, buds, young roots, amenti, the internal bark of roots, the bark of young branches, seeds or other embrionary tissues of vegetals found in the growing phase. These gemma and young branches which come from mature tissues containe many active principles which are not present at the adult plant.

The gemoderivates don't show any intrinsic and extrinsical toxicity, they are easily administrated. They can be individually prescribed or in combination as well for children as the pregnant women, because no side effects were recorded; they can be associated during the treatment with other gemoderivates for obtaining a more synergistically and complex therapeutical result. The gemoderavtes permit the limiting of chemical synthesis drugs, which despite theyre effectivness, they lead to the appearance of side effects.

While the classic phytotherapy uses adult tissues of medical plants for thrapeutical purpose, the gemotherapy uses for the same end only meristematic tissues. Thus in meristemotherapy the following tissues are being used:

The ament: it is also called simply ear, is an inflorescence of reduced sizes, the flowers which compose the inflorescence are unisexual. In gemotherapy only Betula pubescens (downy birch) and Salix alba (willow) inflorescences are used.

The bark of young branches: the internal part of the bark, called part of transition is formed by a layer of living and undifferentiated cells which presenting meristematic cells which produce nothing towards the outside and wood towards the inside. The Citrus limonum and Salix alba bark is used.

Buds: are the vegetative organs of plants and are made of meristematic tissue; thus they are the main tank of primary meristem. The buds are groups of young cells with high nucleo-cytoplasmatic ratio. The buds from the following plants are used: Betula pubescens, Carpinus betulus, Citrus limonum, Ficus carica, Vitis vinifera, etc.

Young branches: they appear through the development of a bud and at they're tip another bud is found. In gemotherapy the shoots from plants at which the harvesting of buds is imposible are used: Buxus sempervires, Rosmarinus officinalis, Jenuperus communis, etc.

Sap: is the liquid which transports nutrients inside the plants, it is formed of the elaborated sap and the enriched sap. Although the sap is not a gemoderivate, its usage in gemotherapy is owed to the fact that it is a dynamic, rich and vital element whose substances have therapeutical propreties. The harvesting of sap in different periods leads to obtaining a substance with different medical propreties. Gemotherapy currently uses only 2 kinds of sap: from birch and willow.

Young roots: root apex, as weel as annex and side roots are rich in meristematic cells. The young roots and the interior bark of roots are rich in meristematic tissues; thus they are used in gemotherapy: the young roots of: Betula pubescens, Secale cereale, Vitis vinifera, Zea mays, etc; and the bark from roots of: Betula pubescens, Vitis vinifera, Quercus peduncolata, etc.

These meristematic tissues are rich in enzymes, growing factors, vitamins, protein, amino, nucleic acids, micropolipeptids, auxine, gibberellinic, cinetina and vegetal hormons.

Gemotherapy from mushrooms primordia

Mushrooms represent a numerous and divers group of organisms which have a unique way of life. They don't have assimilatory pigments and lead a heterotrophic life (saprophytic or parasitic).

The saprophytic mushrooms obtain the necesary substances for life through the decomposing of dead organic material and have a very important role in the substance cycle in nature.

Numerous mushrooms are parasits on plants, animals and on humans. This explaines the practical importance of their study and knowledge.

The mushrooms I used for the preparation of gemoterapics are:

Agaricus bisporus: better known as champignon is very known, apreciated and widely marketed world wide. The hat is white of about 5-10 cm diameter. In the inferior part of the hat, a layer of radial blades is found which form bazidii with 2 bazidiospori.

Posible clinical indications:

It is used for neutralizing the compounds which cause bad breath straight from the intestine having a detoxifying action and it prevents colon cancer.

In the cancer pathology, in particular breast and prostate cancer, hormonorezistent and hormonosensible.

Vitroretinopatia

Helicobacter pylori infection

Kidney failure

Hipercolesterolemie

Alergy pathology

Pleurotus ostreatus: has a brown - grey color and will fade as it grows older. It's hat has 5-15 cm diameter, it is horizontal in shape of a shell or ear, it is fleshy, smooth, with it's edge twisted. The blades are white, the foot is cylindrical, white with 1-2 cm thick and 2-4 cm height.

Posible clinical indications:

Hipercolesterolemia

Lipids metabolism alteration

Atherosclerosis

Prostate cancer

Cardiovascular pathology

Lentinus edodes: it is known world wide as Shiitake. It has a globular hat when it is young and flatten when it reaches maturity. The hat has 5-20 cm in diameter and has a dark brown color when it is young and light brown with small white dots at maturity. The foot is 4-10 cm long.

Posible clinical indications:

Hipercolesterolemia

Obesity

Atherosclerosis

Arthrosis

Viral diseases

Flu

Rhinitis

Liver steathosis

Dental caries

Dental plaque

Cardiac arrhythmia

Stress and it's consequences

Treatment of chemotherapy effects

Cancer: lungs, breasts, leukemia, sarcoma

Impotence.

Chapter II. Materials, methods and results

2.1. The preparation of gemoterapics from primordial mushrooms

Agaricus bisporus

For the preparation of gemoderivates from Agaricus bisporus mushrooms I used a quantity of 65,8203g primordial, from which 15,8203g I used for the dry substance determination and 50g of buds were used for the Gemotherapic. The dried vegetal material is 1,0505g.

The calculation formulas for the obtaining of gemoderivates are:

For the dry substance:

%dry substance=(dried vegetal material/fresh vegetal material)×100

(1,0505/15,8203)×100=6,64%

Dehydrated plant mass:

Vegetal material used for the gemoderivate×%dry substance

50×6,64%=3,32g dry vegetal material

The obtaining of water from buds:

Fresh vegetal material-dry vegetal material

50-3,32=46,68 ml H₂O

Glycerin macerate mass that we want to obtain:

Dehydrated plant mass×20

3,32×20=66,4 ml

The obtaining of alcohol and glycerin volumes necessary for the gemoderivate, using the correction factors of the alcohol (1,25) and of the glycerin(0,81):

(macerated glycerin mass-water from buds)/2

(66,4-46,68)/2=9,86 ml

9,86×1,25=12,325 ml alcohol

9,86×0,81=7,99ml glycerin

Pleurotus ostreatus

For obtaining the macerated glycerin from these mushrooms I used a quantity of 60,1914g primordial from which 10,1914g were used for dry substance and the rest of 50 g fresh vegetal material is used for the gemoderivate.

The calculation formulas for the obtaining of gemoderivates are:

For the dry substance:

%dry substance=(dried vegetal material/fresh vegetal material)×100

(1,0694/10,1914)×100=10,4931%

Dehydrated plant mass:

Vegetal material used for the gemoderivate×%dry substance

50×10,4931%=5,2466g dry vegetal material

The obtaining of water from buds:

Fresh vegetal material-dry vegetal material

50-5,2466=44,7534 ml H₂O

Glycerin macerate mass that we want to obtain:

Dehydrated plant mass×20

5,2466×20=104,932 ml

The obtaining of alcohol and glycerin volumes necessary for the gemoderivate, using the correction factors of the alcohol (1,25) and of the glycerin(0,81):

(macerated glycerin mass-water from buds)/2

(104,932-44,7534)/2=30,0893 ml

30,0893×1,25=37,6116 ml alcohol

30,0893×0,81=24,3723 ml glycerin

Lentinus edodes

For obtaining the macerated glycerin from these mushrooms I used a quantity of 81,0224g primordial from which 2,0224g were used for dry substance and the rest of 79 g fresh vegetal material is used for the gemoderivate.

The calculation formulas for the obtaining of gemoderivates are:

For the dry substance:

%dry substance=(dried vegetal material/fresh vegetal material)×100

(0,3023/2,0224)×100=14,9475%

Dehydrated plant mass:

Vegetal material used for the gemoderivate×%dry substance

79×14,9475%=11,8085g dry vegetal material

The obtaining of water from buds:

Fresh vegetal material-dry vegetal material

79-11,8085=67,1915 ml H₂O

Glycerin macerate mass that we want to obtain:

Dehydrated plant mass×20

11,8085×20=236,17 ml

The obtaining of alcohol and glycerin volumes necessary for the gemoderivate, using the correction factors of the alcohol (1,25) and of the glycerin(0,81):

(macerated glycerin mass-water from buds)/2

(236,17-67,1915)/2=84,4892 ml

84,4892×1,25=105,6115 ml alcohol

84,4892×0,81=68,4362ml glycerin

It is left to be macerated for 21 days at the room temperature, in dark colored recipients, hidden from the direct action of sun rays, being shaken light every day for the whole period.

After the ending of this maceration period we pass to the decantation and filtering of the mixture. The residue after the process is pressed with a steady pressure of approximately 7-10 Pascal. The liquid resulted after the pressing added to the filtrate and is left to rest another 48 hours and then it is filtered again. Thus the base glycerin macerate is obtained from which thru a opportune dilution we obtain the ready to use product.

The dilution: the base glycerin macerate is diluted in proportion of 1:10 with a water-alcohol-glycerin mixture. This mixture is made of 50 mass parts of glycerin, 30 alcohol parts, 20 water parts, thus the first decimal dilution macerate hahnemannian(1DH) homeopath is obtained.

Conservation: the gemoderivates are kept in dark colored recipients, sealed shut and hidden from light. No plastic recipients are used.

The validity term is of about 5 years from the preparation date.

2.2. Total organic carbon analysis from water

Total organic carbon (TOC) is the amount of carbon bound in an organic compound and is often used as a non-specific indicator of water quality or cleanliness of pharmaceutical manufacturing equipment.

A typical analysis for TOC measures both the total carbon present as well as the so called 'inorganic carbon' (IC), the latter representing the content of dissolved carbon dioxide and carbonic acid salts. Subtracting the inorganic carbon from the total carbon yields TOC. Another common variant of TOC analysis involves removing the IC portion first and then measuring the leftover carbon. This method involves purging an acidified sample with carbon-free air or nitrogen prior to measurement, and so is more accurately called non-purgeable organic carbon (NPOC).

Prior to the NPOC determination, the sample must be acidified so that the carbonic acid ions transform into dissolved CO2. This is purged from the aqueous solution (it can be revealed if it can be analyzed by an detector that permits the determination of TIC). After eliminating the inorganic carbon, the organic carbon contained in the pretreated water sample is oxidized becoming CO2 which is revealed by the detector afterwards. The mass of the gas is directly proportional with the total organic carbon (NPOC) contained by the sample

For the water analysis used in the pharmaceutical industry I determined the organic carbon content using the TOC method. I injected 0.5ml of water in the apparatus.

The water used for injection must be pure(sterile)

The furnace temperature must be at about 800 degrees celsius.

For a more accurate result 3 to 5 replicates are made.

The non purgeable organic carbon must be less than 0,500 mg/l.

2.3. Thin layer chromatography identification of botanic species

Thin layer chromatography (TLC) is a method for identifying substances and testing the purity of compounds. TLC is a useful technique because it is relatively quick and requires small quantities of material

Separations in TLC involve distributing a mixture of two or more substances between a stationary phase and a mobile phase. The stationary phase is a thin layer of adsorbent (usually silica gel or alumina) coated on a plate. The mobile phase is a developing liquid which travels up the stationary phase, carrying the samples with it. Components of the samples will separate on the stationary phase according to how much they adsorb on the stationary phase versus how much they dissolve in the mobile phase. To a jar with a tight-fitting lid add enough of the appropriate developing liquid so that it is 0.5 to 1 cm deep in the bottom of the jar.

Separation of compounds is based on the competition of the solute and the mobile phase for binding places on the stationary phase. For instance, if normal phase silica gel is used as the stationary phase it can be considered polar. Given two compounds which differ in polarity, the more polar compound has a stronger interaction with the silica and is therefore more capable to dispel the mobile phase from the binding places. Consequently, the less polar compound moves higher up the plate (resulting in a higher Rf value). If the mobile phase is changed to a more polar solvent or mixture of solvents, it is more capable of dispelling solutes from the silica binding places and all compounds on the TLC plate will move higher up the plate. It is commonly said that 'strong' solvents (elutants) push the analyzed compounds up the plate, while 'weak' elutants barely move them. The order of strength/weakness depends on the coating (stationary phase) of the TLC plate. For silica gel coated TLC plates, the elutant strength increases in the following order: Perfluoroalkane (weakest), Hexane, Pentane, Carbon tetrachloride, Benzene/Toluene, Dichloromethane, Diethyl ether, Ethylacetate, Acetonitrile, Acetone, 2-Propanol/n-Butanol, Water, Methanol, Triethylamine, Acetic acid, Formic acid (strongest).

Working method:

I used 1 g of vegetal material adding over it 10ml of ethanol. After which it is heated on water bath for 15 minutes at 60 degrees Celsius. After cooling the sample is filtered using a small pores micro filter. It will be pipetted 20µl of the filtered solution on the silica gel coated TLC plates.

I used this method for plant identification.

Elutant preparation for the TLC's exposed further below:

11ml formic acid, 11ml acetic acid, 27ml water, 100ml ethyl acetate

Rhamnus frangula: 100 ml ethyl acetate, 17ml methanol, 13ml water.

11ml formic acid, 11ml acetic acid, 27ml water, 100ml ethyl acetate

10ml water, 10ml acetic acid, 40ml buthanol

2.4. Gas chromatography - mass spectrometry

Gas chromatography-mass spectrometry (GC-MS) is a method that combines the features of gas-liquid chromatography and mass spectrometry to identify different substances within a test sample. Applications of GC-MS include drug detection, fire investigation, environmental analysis, explosives investigation, and identification of unknown samples. GC/MS can also be used in airport security to detect substances in luggage or on human beings. Additionally, it can identify trace elements in materials that were previously thought to have disintegrated beyond identification.

Foods and beverages contain numerous aromatic compounds, some naturally present in the raw materials and some forming during processing. GC-MS is extensively used for the analysis of these compounds which include esters, fatty acids, alcohols, aldehydes, essential oils, terpenes etc. It is also used to detect and measure contaminants from spoilage or adulteration which may be harmful and which is often controlled by governmental agencies, for example pesticides.

The GC-MS is composed of two major building blocks: the gas chromatograph and the mass spectrometer. The gas chromatograph utilizes a capillary column which depends on the column's dimensions (length, diameter, film thickness) as well as the phase properties (e.g. 5% phenyl polysiloxane). The difference in the chemical properties between different molecules in a mixture will separate the molecules as the sample travels the length of the column. The molecules take different amounts of time (called the retention time) to come out of (elute from) the gas chromatograph, and this allows the mass spectrometer downstream to capture, ionize, accelerate, deflect, and detect the ionized molecules separately. The mass spectrometer does this by breaking each molecule into ionized fragments and detecting these fragments using their mass to charge ratio.

These two components, used together, allow a much finer degree of substance identification than either unit used separately. It is not possible to make an accurate identification of a particular molecule by gas chromatography or mass spectrometry alone. The mass spectrometry process normally requires a very pure sample while gas chromatography using a traditional detector (e.g. Flame Ionization Detector) detects multiple molecules that happen to take the same amount of time to travel through the column (i.e. have the same retention time) which results in two or more molecules to co-elute. Sometimes two different molecules can also have a similar pattern of ionized fragments in a mass spectrometer (mass spectrum). Combining the two processes makes it extremely unlikely that two different molecules will behave in the same way in both a gas chromatograph and a mass spectrometer. Therefore when an identifying mass spectrum appears at a characteristic retention time in a GC-MS analysis, it typically lends to increased certainty that the analyte of interest is in the sample.

An essential oil is a concentrated, hydrophobic liquid containing volatile aroma compounds from plants. Essential oils are also known as volatile, ethereal oils or aetherolea, or simply as the 'oil of' the plant from which they were extracted, such as oil of clove. An oil is 'essential' in the sense that it carries a distinctive scent, or essence, of the plant. Essential oils do not as a group need to have any specific chemical properties in common, beyond conveying characteristic fragrances.

Essential oils are generally extracted by distillation. Other processes include expression, or solvent extraction. They are used in perfumes, cosmetics, soap and other products, for flavoring food and drink, and for scenting incense and household cleaning products.

Various essential oils have been used medicinally at different periods in history. Medical application proposed by those who sell medicinal oils range from skin treatments to remedies for cancer, and are often based on historical use of these oils for these purposes. Such claims are now subject to regulation in most countries, and have grown more vague to stay within these regulations.

Interest in essential oils has revived in recent decades with the popularity of aromatherapy, a branch of alternative medicine which claims that the specific aromas carried by essential oils have curative effects. Oils are volatilized or diluted in a carrier oil and used in massage, diffused in the air by a nebulizer or by heating over a candle flame, or burned as incense, for example.

The techniques and methods first used to produce Ethereal oil (Essential oil) was first mentioned by Ibn al-Baitar (1188-1248), an Andalusian physician, pharmacist and chemist.

For essential oils contained in citrics (lemon and bergamot) determination I used the gas chromatography-mass spectrometry. I diluted 1:100 the test sample with dichloromethane, I injected 2 micro liter from the prepared solution in the GC/MS using the not polar column 2.mth, the whole process took 110min.

The GC/MS work condition are:

Injection temperature: 250 degrees Celsius

Oven temperature: 120 degrees Celsius

FID temperature: 280 degrees Celsius

Carrier gas: nitrogen or helium

The results are the following:

2.5. High performance liquid chromatography

For the analysis of the vegetal prime materials that can be contaminated with ocratoxin A like coffee, cereal, cocoa and beverages I used the high performance liquid chromatography.

Ochratoxin A, a toxin produced by Aspergillus ochraceus and Penicillium verrucosum, is one of the most abundant food-contaminating mycotoxins in the world. Human exposure occurs mainly through consumption of improperly stored food products, particularly contaminated grain and pork products, as well as coffee, wine grapes and dried grapes. The toxin has been found in the tissues and organs of animals, including human blood and breast milk. Ochratoxin A toxicity has large species- and sex-specific differences.

Synonyms:

• (R)-N- [(5-Chloro- 3,4-dihydro- 8-hydroxy- 3-methyl- 1-oxo- 1H-2-benzopyran-7-yl) -carbonyl]- L- phenylalanine
• (-)-N- [(5-Chloro- 8-hydroxy- 3-methyl- 1-oxo- 7-isochromanyl) carbonyl]- 3-phenylalanine

Ochratoxin A is potentially carcinogenic to humans (Group 2B). Ochratoxin A has been shown to be weakly mutagenic, possibly by induction of oxidative DNA damage.

There is sufficient evidence in experimental animals for the carcinogenicity of ochratoxin A. Ochratoxin A was tested for carcinogenicity by oral administration in mice and rats. It increased the incidence of hepato-cellular (Hepatic tumor) tumours in mice of each sex and produced renal-cell adenomas and carcinomas in male mice and in rats of each sex.

High-performance liquid chromatography (or high-pressure liquid chromatography, HPLC) is a chromatographic technique that can separate a mixture of compounds, and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.

HPLC utilizes different types of stationary phase (typically, hydrophobic saturated carbon chains), a pump that moves the mobile phase(s) and analyte through the column, and a detector that provides a characteristic retention time for the analyte. The detector may also provide other characteristic information (i.e. UV/Vis spectroscopic data for analyte if so equipped). Analyte retention time varies depending on the strength of its interactions with the stationary phase, the ratio/composition of solvent(s) used, and the flow rate of the mobile phase.

With HPLC, a pump (rather than gravity) provides the higher pressure required to propel the mobile phase and analyte through the densely packed column. The increased density arises from smaller particle sizes. This allows for a better separation on columns of shorter length when compared to ordinary column chromatography.

The reagents I used for the determination of ocratoxin A are: phosphoric acid 0.1M, chloroform, celite545, aqueous solution of sodium bicarbonate 3%, methanol, distilled water, solution: ethyl acetate: methanol: acetic acid (95:5:0.5), ethyl acetate, mobile phase(HPLC) water: acetonitrile: acetic acid (99:99:2), ocratoxin A OEKANAL analytical standard 5mg.

Extraction phase: I weighed 50g of sample, I added 250ml chloroform and 25 ml phosphoric acid 0.1M, it was extracted for 3 minutes at high speed. Before the ending of the phase 10g of celite545 is added. I filtered an gathered 50 ml of the filtered substance and I transferred it to a separation funnel, I added 10ml of aqueous substance sodium bicarbonate 3% over it. After I shook it, I left it alone for the two phases to separate. The superior aqueous phase (EST1) is gathered and is passed on in the column.

Purification phase: The C-18 column (Supelco, Discovery DSC-18 SPE Tube) is tied to void equipment for the extraction in the solid phase. The column is conditioned 2 times with 2 ml of methanol, 2 ml of distilled water and 2ml of aqueous solution of sodium bicarbonate 3%. For avoiding the dry out of the column 2 ml of solvent is assured to always be present on the frit, during this phase. 5 ml of EST1 is passed through the column, after which the column is washed with 2ml phosphoric acid 0.1M and 2ml of distilled water, the washing process is stopped. Ocratoxin A is eluted with 8ml ethyl acetate: methanol: acetic acid (95:5:0.5) and it is collected into a stoppered tube which already contains 2 ml of distilled water.

Final extraction: I separated the aqueous phase from the organic phase. From the aqueous phase I made 2 extractions with 1 ml of ethyl acetate each time. The organic phases are mixed and passed thru a nitrogen current. The sample is treated with 1ml of mobile phase. The sample substance is thus prepared to be injected in the HPLC.

The labeling curve for Ochratoxin A is:

Ochratoxin A determination in the vegetal material sample of Glycyrrhiza glabra with known content of Ochratoxin A is:

2.6. Atomic absorption spectroscopy

In analytical chemistry, atomic absorption spectroscopy is a technique used to determine the concentration of a specific metal element in a sample. The technique can be used to analyze the concentration of over 70 different metals in a solution.

In order to analyze a sample for its atomic constituents, it has to be atomized. The sample should then be illuminated by light. The light transmitted is finally measured by a detector. In order to reduce the effect of emission from the atomizer (e.g. the black body radiation) or the environment, a spectrometer is normally used between the atomizer and the detector

For phisico-chemical analysis I used the atomical absorption spectrophotometer (SpectrA A). The vegetal material sample was prepared as following: oxygenated water, nitric acid, sulfuric acid were added over 1g of vegetal material. The sample was heated until the solution was clarified. The so obtained solution was diluted with acidified water in report of 1:100. The distilled water was acidified with nitric acid 0.5%. From this sample I determined the heavy metals(Cd, Pb, Hg) and arsenic using the SpectrA A.

For Cd determination, the work conditions are:

The measurement unit was µg/l

It was read at 228,8 nm

The lamp was on position 3

The lamp intensity is of 10,0 mA

3 standard replicas are made

For the etalonation curve I used the standard of Cd 0,500 µg/l, 1,00µg/l, 1,500µg/l

The atomizer used on this atomic absorption was an graphite furnace

Cd is read at 1800 degrees Celsius

For Arsenic determination the working conditions are:

The measurement unit was µg/l

The atomizer used on this atomic absorption was an graphite furnace

3 standard replicas are made

For the etalonation curve I used the standard of Arsenic 25,00µg/l; 50,00 µg/l; 75,00µg/l

It was read at 193,7 nm wave length

Lamp position is 4

The lamp intensity is of 10,0 mA

It was read at 2600 degrees Celsius

For Pb determination, the working conditions are:

The measurement unit was µg/l

The atomizer used on this atomic absorption was an graphite furnace

3 standard replicas are made

For the etalonation curve I used the standard of Pb 15,00µg/l; 30,00µg/l; 45,00µg/l

It was read at 283,3nm wave length

The lamp intensity is of 10,0 mA

The lamp position is 3

It was read at 2100 degrees Celsius

For Hg determination, the working conditions are:

The measurement unit was µg/l

The atomizer used on this atomic absorption was hydrides

3 standard replicas are made

For the etalonation curve I used the standard of Hg 10,00µg/l; 20,00µg/l; 40,00µg/l

The lamp intensity is of 4,0mA

It was read at 253,7nm wave length

The lamp position is 2

The measurement time is 5,0 s

Hydrides type: cold vapors

The results obtained on different types of vegetal material are:

2.7. In vitro cultures

I have also made some research concerning cultivation of Crocus sativus in vitro.

The history of saffron cultivation reaches back more than 3,000 years. The wild precursor of domesticated saffron crocus was Crocus cartwrightianus. Human cultivators bred wild specimens by selecting for unusually long stigmas. Thus, a sterile mutant form of C. cartwrightianus, C. sativus, emerged in late Bronze Age Crete. Experts believe saffron was first documented in a 7th century BC Assyrian botanical reference compiled under Ashurbanipal. Documentation of saffron's use over the span of 4,000 years in the treatment of some 90 illnesses has been uncovered.

The domesticated saffron crocus (C. sativus) is an autumn-flowering perennial plant unknown in the wild. It is a sterile triploid form, possibly of the eastern Mediterranean autumn-flowering Crocus cartwrightianus that originated in Central Asia. The saffron crocus resulted when C. cartwrightianus was subjected to extensive artificial selection by growers seeking longer stigmas. Being sterile, the plant's purple flowers fail to produce viable seeds; reproduction depends on human assistance: corms, underground bulb-like starch-storing organs, must be dug up, broken apart, and replanted. A corm survives for one season, reproducing via this division into up to ten 'cormlets' that yield new plants. Corms are small brown globules up to 4.5 centimetres (1.8 in) in diameter and are shrouded in a dense mat of parallel fibers.

Stigma-like structures (TC stigmas) were produced in tissue cultures from stigma explants of Crocus sativus under defined conditions. MS medium supplemented with NAA (10 mg dm^-3)+ BA (1.0 mg dm^-3) induced the optimum response. NAA was found to be an important addendum to achieve a good response. A culture temperature of 20 °C seems to be better than 25 °C with reference to all parameters.

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