Isolation, Identification and characterization of specific chemical compound marker of 1000.8 KD in shilajit sample responsible for the therapeutic activity of shilajit prescribed in Ayurved as panacea.
[0001]Technical Field: This invention relates to the field of Ayurved and nurtiien supplementsgenerally and more specifically to the isolation identification and
characterization methods for the differentiation between the pure Shilajit and ajulterated
Shilajit with ordinary humus contents.
|()0021BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to extraction, isolation, identification and characterization of specific chemical compound from crude Shilajit and further investigation of chemical compound for its therapeutic efficacy. The other spotlight of the present invention is to highlight the fact that the therapeutic activity of shilajit is not only due to the presence of 1 lumic acid, Fulvic acid and alpha-pyrons.
[0003]BACKGROUND :- Shilajit has been considered to restore life and considered to have miraculous powers . Shilajit has been considered in orient to virtually cure an\ ailments and make the human body rock like being able to withstand the ravages of time.
[0004]The greatest Ayurvedic physicians have said "There is no disease on the earth which can not be cured by Shilajit"". Shilajit has been considered to restore life and considered to have miraculous powers . Shilajit has been used as a rejuvenator and an adaptogen for thousands of years.

[0005]Myth about the discovery of Shilajit; White monkeys was reported to consume the semi-soft exude in the layers of rock in warm summer months, villagers attributed this to the strength, longevity & wisdom of the monkeys and they also starts consuming the exude material. The name Shilajit was derived from Sanskrit word (SH1LA= ROCK. JIT TRIUMPH). Shilajit is a symbol of energy and supposed to cure all the disea.ses since centuries in India.
[0006]Prior Art: - Shilajit is a pale-brown exudation of variable consistency, oozing out from layer of rocks in many mountain ranges of the world. It originates from bioorganic material. It is important to know what chemicals in Shilajit bring about such a power. Prier research has been suggested that physiological action of shilajit has been attributed due to the presence of bioactive dibenzo-alpha-pyrons alongwith humic and fulvic acids. Fulvic acids: - Fulvic acid is a water-soluble natural organic substance which is derived from humus. It was known as a tremendously complex mixture of low molecular weight organic compounds. Fulvic acid is also occurred naturally in organic plants and soils.
[0007]Early work on shilajit showed that it is mainly composed of humus - the characteristic organic constituent of soils - together with other organic components. Humus consists of organic residues that have lost their original structure following rapid decomposition in the environment. It was not a clearly defined substance earlier .The composition of shilajit could changes constantly and it can disappear b> slow decomposition unless new residual matter is incorporated. Humus contains two components of interest: fulvic acid and humic acid. The former has the lowest molecular weight components, containing uronic acids, phenolic glucosides and amino acids, while the latter is composed of high molecular weight compounds and contains a high proportion of phenolic.

[0008]The humus extracts from shilajit were shown to possess a lattice-like structure, perforated by voids of varying dimensions (0.010.05 mm). These are filled with organic molecules or metal complexes, which may be responsible for the therapeutic effects.
[0009]Chemical research in the 1970s indicated that shilajit is quite recent in origin. In 1976. components of the plant Euphorbia royleana were discovered in shilajit. making the ancient fossil route unlikely. The plant is named after Euphorbias, who was the court physician of Juba II - the Romanised ruler of a North African kingdom - in the first century AD. There are cu 100 species of this plant growing wild in most temperate regions of the world. When damaged, they exude milky latex that irritates the skin and eyes. The latex was considered to be transformed into the components of shilajit.
[0010]Some of the organic compounds in Euphorbia royleana and shilajit are also found in animals such as beavers, which frequently eat the buds and bark of trees, and this is thought to be responsible for the deposits in these animals.

(Figure Removed)
Fig 1. Some of the compounds reported to be present in shilajit
[0011]However, the controversy about the origin of shilajit raged on until the mid-1980s, when clear evidence emerged, indicating that the substance was of recent plant origin. Chemical analysis of shilajit by researchers at Banaras Hindu University in India revealed that, among other compounds, it contains biphenyl metabolites. (Fig 1). Interestingly, the group also identified some of the same biphenyls (13) in Trifolium repens. a plant that grows abundantly in regions where shilajit is found. This led to the conclusion that humification of some resin/latex bearing plants is the most likely source of shilajit.

When Banaras researchers subjected compounds (2) and (3) to column chromatography. they discovered that they were partially transformed to a benzocoumarin (4). Ihis compound (4) has also been detected in shilajit, possibly arising via an intermediate compound (5). The results were quite exciting because biphenyls are a rare group of natural products in higher plants, and no biphenyl with a carboxy function at C6 had been encountered before. They reported that, compounds (2), (3) and (4) have significant antiallergic activity - a property claimed for shilajit.
[0012]By the late 1980s it was known that humification of resin-bearing plants was responsible for the major organic mass of shilajit - about 80 per cent of the humus component. The amount and composition of the remaining organic mass, which is a mixture of low M„ compounds, varies depending on where the shilajit comes from (Table 1). The most common low M„ compounds present are oxygenated dibenzo-a-pyrones (6). (7) and (8). They are probably produced from the naturally-occurring macrocycle (9). via an intermediate such as (10), (Scheme I). Solvent extraction of shilajit results in a compound similar to (10), which can be autoxidised to (6) and (8) when exposed to light.
[0013]The all the reported researchers proposed that the physiological properties of shilajit are due to compounds such as the dibenzo-a-pyrones. along with triterpenes and phenolic lipids. Fulvic acids may also have a physiological role, acting as carrier molecules for the more bioactive smaller compounds.
(Scheme Removed)
Scheme 1. A possible route to making dibenzo-a-pyrones (6) and (8)

[0014)Anwher, K et. al. 2007 reported the direct elemental composition analysis of complex mixture of Shilajit by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. Amir et. al. 2006 reported the structural characterization of fulvic acids, extracted from sewage sludge during composting, by thermo chemoiysis-gas chromatography-mass spectrometry.
[0015]They were applied the thermo chemolysis coupled with gas chromatography and mass spectrometry and determine the chemical structure of fulvic acids (FA) extracted from a sewage sludge and straw mixture at different steps of composting. The FA starting structures were composed mainly of mono-, di- and tri-methoxy (alkyl) benzoic acids representing lignin derivatives in an advanced stage of oxidation, as well as of methylated polyphenols and a series of fatty acids saturated C15. C16, C18. unsaturated and branched C18:1. Besides, there are many unidentified structures suspected to be N-containing compounds. During the composting process, the fate of the three structural typical monomer units of lignin was followed. The p-hydroxyphenyl units showed a strong relative decrease especially at the beginning of composting. The guiaicyl units showed a steady increase in course of composting. A relative decrease of syringyl units was noted at the beginning of composting, but they then relatively increased towards the end of composting. These changes support the formation during composting of more oxidized units. All subunits composing the fulvic acid structures have been subdivided into main five groups of similar chemical structure. The lignin-like C6-C3 subunits showed a relative decrease during composting attributed to microbial oxidation, but there was a relative increase during the intermediate step of composting related probably of an enhancing of lignin solubility.
[0016] The C6-C1 subunits were reduced at the beginning of composting, which mainly attributed to the oxidation of 4-methoxybenzaldehyde to hydroquinones derivatives under the composting conditions. Although, the relative increase of these subunits (C6-C1) at the end of composting originates from oxidation of C6-C3 lignin side-chains or could be partly attributed to microbial neosynthesis. The unidentified N-containing compounds increased strongly during course of composting. The rise in the level of fatty acids at the beginning of composting is attributed to an increase of branched-chain fatty acids such as C18:1 commonly used as bacterial biomarkers. Their amounts were greatly reduced at the

end of composting. Both the Shannon Weaver and simihtude indices show a relative increase in structural diversity at the start of composting conditions following the appearance of hydroquinone derivatives and unidentified nitrogen compounds in the F.A network. But. the produced FA structure reaches a certain level of homogeneity at the end of composting through self-polycondensation or recombination of C6-C1 subunits and hydroquinones derivatives with N-containing compounds.
[0017]Khanna et. al. 2008 reported the Spectroscopic characterization of fulvic acids extracted from the rock exudates Shilajit. They described the extraction of fulvic acids (FA) from Shilajit and its spectroscopic and mass spectrometric characterization. The spectral features obtained from FT-IR and 1HNMR were similar to those reported for humic substances from other sources. The molecular elemental composition analysis by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) in electrospray negative ion mode resulted in extraordinary high average O/C ratios (0.53) which might be caused by a significant contribution of carbohydrates in Shilajit. ,A very high average H/C ratio of 1.27 also points to dominant aliphatic or alicyclic structures and relatively low aromaticity. The average molecular formula of the nitrogen free elemental compositions measured by FT-ICR mass spectrometry is C18.2H23.0O10.0.
[0018]Prior Art for Pharmacology of Shilajit: - Shilajit is a blackish-brown exudation, from steep rocks of different formations, commonly found in the Himalayas, at altitudes between 1000-5000 m, from Arunachal Pradesh in the East to Kashmir in the West. It is also found in other countries, e.g. Afganisthan (Hindukush), Bhutan. China. Nepal. Pakistan, Tibet (Himalayan belt) and the USSR (Tien Shan,Ural). Shilajit is believed to arrest aging and produce rejuvenation (ref. 11, -two important attributes of a ra.sayan (Datta et. al. 1877 and Sharma et. al 1978). This is a very broad statement recent researches have proven that it"s adaptogenic properties can indeed benefit everyone, however there are certain conditions it is especially effective for, like memory, immune system modulator, anti ulcer, anti inflammatory & anti oxidant. It is important to know what in Shilajit brings about such a power. I he active chemicals of Shilajit ensure the transportation and the absorption of fulvic acid by the body cells to the full extent and no residual material remains in the cells. This chelation of ions is probably the most important role of active constituent of shilajit with respect to living systems. Eiy chelating the ions, they facilitate the uptake of these ions by several mechanisms, one of which is

preventing their precipitation; another seems to be a direct and positive influence on their bioavailability.
[0019]Fulvic acid is activation agent of varies of cruor gene. It can be oral hemostasis when the stomach, colon, nephridiuni and womb bleed. It can also improve cycle obstacle of the heart, brain and stomach, reduce the viscidity degree of blood plasma and blood circulation, has unique operation of activating blood circulation and dispersing blood clots. It can be used for clinical treatment of gastric ulcer, colonitis. mastitis, sugar diabetes, sucker liver and cancer acesodyne etc. Ghosal et. al. 1990 reported the chemistry and immunomodulatory action of Shilajit. They concluded that 80-85% of major organic mass of shilajit was a humification of latex and resin-bearing plants. The low mol. wt. chemical markers (8 to 10%). viz. aucuparins. oxygenated dibenzo-K -pyrones and triterpenic acids of the tirucallane type (free and conjugated), occurring in the core structure of shilajit humus, was the major active constituents of Himalayan shilajit.
[0020|Clinical applications of shilajit in Ayurveda, as a rasayan. are well documented {refs.1,3). However, no modern scientific study was carried out before on the mode of action of shilajit. The effects of shilajit. as reported in the Ayurvedic literature, seem to suggest its influence on endocrine, autonomic, and brain functional changes. The discovery that these changes can be mediated by cytokines, released by activated immunologic cells (ref 11>, has opened up possibilities for similar mechanism of action FAs of shilajit produced significant effects against restraint stress-induced ulcers (ref 6).The mechanism of artti-ulcerogenic actions of shilajit and its constituents was also evaluated (ref6). This was based on their effects on mucin contents, and on the concentrations of DNA and protein in the gastric juice.The combinations providedsignificant resistance to mucosa against the effects of ulcerogens and also prevented the shedding of mucosal cells. The anti-allergic action of these compounds was

successfully tested against antigen- and compound 48/80 (histamine releaser)- induced de granulation of mast cells (ref 12).
[0021]The anti-stress activity of these compounds was suggested by their augmentation of murine swimming endurance exercises. Shilajit and its combined constituents also elicited and activated, in different degrees, murine peritonealmacrophages and activated splenocytes of tumour-bearing animals at early and later stages(unresponsive) of tumour growth (tested according to ref 13).
Shilajit from USSR, and its corresponding combined fractions, acted essentially as cell-growth factors in both normal and tumour cells by maintaining membrane integrity. The results obtained till now are sufficiently impressive to warrant expectation that more extensive and comprehensive studies on shilajit and its constituents would validate the Ayurvedic rasayan. shilajit,as more effective than several currently available clinically efficacious immunomodulators (refs. 14. 15). Kanikkannan et. al 2006 has reported the shilajit-induced potentiation of the hypoglycaemic action of insulin and inhibition of slreptozotocin induced diabetes in rat. They repoted the effects of subcutaneous (s.c.) administration of processed shilajit (PS) alone and in combination with insulin (s.c.) on plasma glucose levels (PGL) were determined in either sex of strepto/olocin-induced diabetic (SID) rats. PS (50 µ/kg; s.c.) did not alter plasma glucose levels in SID rats. Insulin (0.25-1.0 U/kg; s.c). dose-dependently produced hypoglycaemia in SID rats. PS (50 µg/kg: s.c.) potentiated and prolonged the hypoglycaemic action of insulin when administered concurrently. Chronic administration of PS (0.1-10 mg/kg: b.i.d. for 10 days; i.p.) had no influence per se on plasma glucose levels. Chronic administration of PS (1.0 mg/kg; b.i.d. for 10 days; i.p.) prevented the streptozotocin (STZ)-induced diabetes in rats, whereas a relatively lower (0.1 mg/kg; b.i.d. for 10 days; i.p.) and significantly higher dose (10 mg/kg; b.i.d. for 10 days; i.p.) of PS had no influence on the S fZ-induced diabetes. Their results suggest that administration of PS along with insulin would potentiate the insulin-induced hypoglycaemia. and chronic administration of a carefully determined dose of PS would inhibit the development of STZ-induced diabetes.

[0022] Properties and action according to the Ayurved
Rasa: tikta, kashaya
Guna': laghu, snigdh
Virya: usna
Vipaka: madhura
Karma: vatakapha hara, balya, rasayana, vajikama.
[0023]Modern therapeutic classification index:
• Blood and haemopoeitic tissue: Iron is required to make red blood cells. Shilajeet is a good source of trace minerals and contains Iron. The fulvic acids help carry the iron into the body making it bio-available.
• Respiratory system: Since it also promotes expectoration, it makes it useful in respiratory illnesses. It may be used in acute and chronic bronchitis. The antispasmodic qualities brings it's effects with asthma
• Reproductive system Shilajeet is an aphrodisiac. It increases the libido of men and is an aphrodisiac
• Nutrition and metabolism: Shilajeet has long been used for the prevention and treatment of diabetes in Ayurvedic medicine
• Genito- urinary system: Shilajeet is used in kidney function. It is effective in treating burning urine, incontinence of urine due to enlarged prostate or stone in the bladder or kidney
• Immunity system: Antioxidants can safely neutralize a free radical without becoming a free radical them self Shilajeet is a powerful antioxidant that has the added benefit of being able to cross the blood-brain barrier
[0024]Need of My Invention: -
in present boom in ayurvedic and herbal products the alarming and emerging problem for manufacture of herbominiral preparation is to preserve the actually pharmacologically active substance for healing properties of herbominiral medicines. Without the presence of actual pharmacologically active substance or presence of other pharmacologically active substance in the herbominiral preparation would not act accurately and even can harm the user. The actual pharmacologically active substance is responsible for specific therapeutic effects on the various systems of humans.

[0025]The growing body of scientific literature from various domains sheds a great deal of light on potential uses for shilajit in human nutrition, in topical applications to the skin, and for the treatment of various diseases. Although much research remains to be done on the subject, early indications are quite favorable to support the supplementation of shilajit in the diet.
[0026]As there has been substantial benefit attributed to the human by the use of shilajit. a need exists in the art for specific marker which could differentiate between the shilajit humus and ordinary humus. We presume that the substantial therapeutic benefit attributed to the human by shilajit is not merely due to the presence of three i.e. Fulvic. Humic and dibenzo-alpha-pyrons. But it is due to the presence of other specific chemical compounds in combination of these three.
[0027|The large market & heavy demand of shilajit the adulteration and substandard shilajit was providing by the suppliers. Shilajit is one of the key ingredients of major Ayurvedic preparations.
[0028]The lack of specific analytical method of identification of shilajit motivate us for the development of the specific method for the isolation, identification and characterization of novel chemical markers for the differentiation of in the shilajit humus and ordinary or adulterated shilajit humus.
[0029]The present invention will provide the right way for adopting the pure Shilajit by using my newly invented scheme and method expressed in this invention which was never available earlier any where in the universe.
A scheme and method for the isolation and identification of 1000.8 KD chemical compound in shilajit and further development of said compound as specific marker for the characterization and differentiation pure shilajit from similar adulterants is obtained via a process of stated in the "METHOD AND MATERIAL"" section of specification.
[0030]Detail description of present analytical method for the isolation, identification and characterization of 1000.8 KD chemical compound.
The main objective of present invention is to study and differentiate the chemical constituents of shilajit humus and ordinary humus samples. The results of our study on various samples of shilajit and different humus samples suggest that the major chemical constituents present in shilajit humus structurally similar to ordinary soil except some chemical entities such as 1000.8 kD chemical compound.

[0031]METHOD AND MATERIAL
The analytical condition for the isolation, identification and characterization of specific marker was following.
Materials
High performance silica gel-precoated thin-layer plates (HPTLC, Kieselgel 60) were obtained from Merck (Darmstadt, Germany). All the chemicals were of the highest purity available.
[0032]Extraction of bioactive chemical fraction from crude shilajit and ordinary humus: - we have taken 10 gm sample of shilajit and ordinary humus, the isolation of substances was performed by the classical method of humus substances fractionation. We have taken 10 gm of each sample in 50 ml of chloroform in reflux apparatus, fitted with a connection tube for nitrogen fluxing, was adapted to the flask and the reaction was allowed to proceed at 90°C under vigorous stirring and reflux the samples for 3 hours. Then we discard the chloroform layer and extract the sample with 50 ml of ethyl acetate and maintain the pH by adding the .01N Na OH . Before further extraction we passed the N2 gas for half an hour, then we discard the ethyl acetate layer and reflux the sample with 50 ml methanol for one hour. Then we washed the residue with 5 volumes of cold acetone and stored overnight at 4°C. The residue was dissolved in the minimum amount of methanol.

Analysis of possible elemental connposition of 1000.8 peak

TOF Mass spectrum showing isotopic distribution of 1000.8
(Figure Removed)
Figure 1. Electron spray ionization mass spectrometry of the pavitra compound prepared from shilajit .The TOF Mass Spectrum 0.015 TO 0.831 min from sample showing isotopic distribution of 1000.8589 compounds.

[0033]According to certain embodiments of the present invention, the 1000.8589 kD compounds may be purified or substantially purified before incorporation into a composition for human use. In certain embodiments, the l000.8589 kD compounds may be that obtained via purification using Preparative HPLC other embodiments of the present invention, the 1000.8589 kD chemical compound can only be isolated from the humus of crude shilajit. Further embodiments of the present invention, the 1000.8589 kD chemical compounds is not isolated from ordinary humus. River water, or coal.
[0034]According to one embodiment of the present invention, a composition for human use containing 1000.8589 kD chemical compound is provided. The composition comprises at least 700 mg/L fulvic acid and preferably between 700 and 20,000 mg/L. fulvic acid. Exemplary forms of these compositions include, but are not limited to, tablets, capsules, granules, supplements, foods, creams, ointments, emulsions, suspensions, liquids, drinks, beverages, sprays, inhalers, suppositories, drops, cosmetics, or other forms designed to deliver fulvic acid to a subject. The composition for human use may be in the form of a cosmetic. Such cosmetics are exemplified by. but not limited to. lotions, milky lotions, creams, facial packs, ointments, tooth pastes, bathing agents, bath detergents, facial cleansing agents.
[0035]Further embodiments of the present invention relate use of fulvic acid in the manufacture of a composition for promoting hair growth. The present invention is further
described in the following example, which are offered by way of illustration and are not
intended to limit the invention in any manner. [0036] EXAMPLES
EXAMPLE 1
Isolation of 1000.8589 kD chemical compound from Crude Shilajit.
We have taken 10 gm sample of shilajit and ordinary humus, the isolation of substances was performed by the classical method of humus substances fractionation. We have taken 10 gm of each sample in 50 ml of chloroform in reflux apparatus, fitted with a connection tube for nitrogen fluxing, was adapted to the flask and the reaction was allowed to proceed at 90°C under vigorous stirring and reflux the samples for 3 hours. Then we discard the chloroform layer and extract the sample with 50 ml of ethyl acetate and

maintain the pH by adding the .01N Na OH . Before further extraction we passed the N2 gas for half an hour, then we discard the ethyl acetate layer and reflux the sample with 50 ml methanol for one hour. Then we washed the residue with 5 volumes of cold acetone and stored overnight at 4°C. The residue was dissolved in the minimum amount of methanol.
EXAMPLE 2
Isolation of bioactive fraction from the extract of shilajit by HPTLC:-HPTLC Details
The Linearity of Detector Response was done by Hamilton syringe, the working standard mixture was spotted as sharp bands on plate with the concentration of 0.025mg/ml(2 ul -6ul) of extracted material. Sample solution: Methanol
Development system: we have tried many solvent systems but ultimately we get best result with Chloroform: Formic acid: Ethanol: Water (9:1:1:. 10).The solvent were allowed to travel a distance up to 80%.
Stationery Phase: we have also tried four stationery phase but ultimately we get best result with normal phase pre coated silica gel 60 F254 TLC plate of 0.2 mm thickness. Sample Application: - 20 µl of each samples was applied with Linomat IV. band length was 20 mm.
Densitometric evaluation: - With Camag TLC scanner and CATS software, evaluation via peak area by linear regression.

EXAMPL-3
The characteristic absorption bands typical for humic and fulvic substances was observed at 357 nm. The chromatographic investigation has revealed the presence of characteristic fluorescent organic matters in extracted fractions. Further we did the cutting of the fluorescent band and dissolved the material in methanol. Then put the test tubes in shaker for night. Then we filter the dissolved material by whatman filter paper no.l.
EXAMPLE- 4
API 4000 Q Trap LC/MS/MS and QSTAR Elite system was found to be the best for the confirmation and identity of fluorescent zone on the HPTLC plate. API 4000 Q Trap LC/MS/MS and QSTAR Elite system was more selective than UV detection.

EXAMPLE-5
LC/MS/MS by QSTAR Elite of isolated fraction
Analytical conditions for LC-MS/MS
Instrumental condition
Mobile phase A: Water
Mobile phase A: Acetonitrile
Flow rate: 0.2 ml ml/min
Column: C18.150 mm X 4.6 mm (5µm)
Gradient:
(Table Removed)
Mass Spectrometer API 4000 (Sequence Removed) Trap LC/MS/MS System
Software Version: Analyst 1.4.1
Scan type: QlMS(Ql)
Polarity: Positive
Ion Sources Turbo Spray

(Table Removed)
EXAMPLE- 6
1000.8589 kD compound named as Pavitra was selected as a specific chemical marker for the identification of shilajit .The 1000.8589 kD compound species was not present in any of the ordinary humus sample. The 1000.8589 kD compound species was responsible for the long wavelength band and presumably by the presence of non-humic substances. We suppose that 1000.8589 kD pavitra compound consists of coumarin derivatives, which are probably responsible for the fluorescence.
EXAMPLE- 7
We have tested this fraction for their ability to modulate reactive oxygen species (ROS) production. Intracellular phorbol-12-myristate-l3-acetate (PMA) stimulating the ROS formation was determined by fluorescence with the use of 2',7'-dichlorofluorescein diacetate. The dose dependent activation in ROS production was observed with increasing concentrations of 1000.8589 kD compound, and duration of cell preincubation with these shilajit constituents. On the contrary. 1000.8589 kD compound significantly suppressed the macrophage activity.
EXAMPLE-8
Measurement of Mitochondrial Membrane Potential in Intact Cells. Cells were incubated at 37°C and 5% CO2 with the appropriate 1000.8589 kD isolated compound from shilajit only. The PTPC inhibitors were added 30 minutes before the addition of methylation of isolated compound. Carbonyl cyanide 3-chlorophenylhydrazone. a carbonyl cyanide that served as a positive control, was added at 10 µmol/L immediaiely before the loading with DiOC6(3). The cell suspension was incubated with 0.4 nmol/L DiOC6(3), a fluorescent probe used to measure mitochondrial membrane potential, until the time of analysis (usually 30 minutes).

EXAMPLE-8
I be findings presented by our inveiMion widest that 1000.8589 kD isolated oxnpuund was mitochondriotoxic townrd human eaaeer ceil lines, in a PTPC-meditUed mechanism.
i ^ WII': I. l!
\ sli; I: p'Hi composition is prepared according to the following recipe: lf09.1 K9 ciMaHcal cempiMMldl la alMajit in an amount to obtain a linal concentration of I .0(MJ U
:0()() mg/L, 15 g Giio\ hydrol>/.ed protein extract. 20 g slinging nettle leaves CMUKI -0 i: (lAMAI. SBS-I 1 (detergent). 20 g
(T \MAI. NO-.) (detergent). 0.4 g aseplina. l.(. g ethanol. 0.04 g BRONOroi, (preser\ative). 6 g sodium chloride. 106 g ol water.
\n alternate shampoo composition is prepared according to the following recipe; fuls \c acid in an amount to obtain a fmal concentration of 1.000 to 20.000 mg/1., 13 g horse chestnut extract, 22 g marigold extract. .U) g GAMAl. SBS-ll
(detergent). 20 g GAMAL NO-3 (detergent), 0.4 g aseptina. 1.6 g ethanol. 0.04 g
UKO\()P()I. (preservative). 6 g sodium chloride. 106 g of.water.
I \ WIIM I- 1 I
, \ loot!) paste comprising 1#MM K9 Aaaiical raipniiad iraaa sMIajit kaatas m
■in amount to obtain a tlnal concentration of 1000 to 2000 mg/L. ethcal oils or ilicir ctMiiposnions or else fruit essences in an amount of l-10"'(i by weight, ghcerol '.i-. ai, inuiiHit (if .■S-l()% by weight. Akarkera herb extracts in an amount of 0.10 10"(i by ue.L'h; ,IIK1 cleaning substances in an amount oi 20-"vS"/b by weight dispersed in water m an .iniouni of 4.5-60% b\ weight, and d\es and whitening components in an amount ol I -2"> hy weight.
i itanium dioxide may be used as a whitening comptment; sage leaves, chamomile or marigold tlowers extracts ma\ be used as beneficial preferred herb extracts.
iXAMPIt i:
A bath salt preparation. 97 g siilt (Nat I) containing occluded fulvic acid in an amouiii U) Mhiain a final concentration o\' 1.000 !>) I'd.000 mg/L and-3 ;.; pine ethereal oil oi- •(•, r.;!! (MIS conipusition is prepared.
1 XAMI'l.f; 1.-^
' oMiietie masks are well known cosmeiic preparations serving many dilfcieiit purj^' .
\ cosmetic mask comprising lMi>Ǥl^ iiwirai cMSpeaiMlMlaWllliit humu^ \u anuiuiit to obtain a final concentration o\' KK) lo ."!(l() mg/L. 20 g natural therapeutic miKi.
U) 2 of humic acid. lO g magnesium caibonaic > ;_■ zinc o\ide. 0.2 g citiric acid. 5 e i