Quality control of SEKEM RENAL 1.5 G

The purpose of report:

-1-

safety

-2-

efficacy-3-

Quality

SEKEM Renal Herbs which is a natural mixture of herbs:

OAmmi visnaga fruits

OCymbopogon leaves

OAmbrosia leaves

OAchillea leaves

Chicory leaves

OPeppermint leaves

OLicorice root.

Indications:

O1-Renal colic

O2-Sandy and renal stones

O3-Nephrolithiasis

O4-Prophylactic against

urinary tract infection

Tea preparation:Put a filter-bag of SEKEM Renal Tea in 150 ml of boiling water. Allow to steep for 3-5 minutes. Drink 2-3 times a day.

Contraindications:Use during pregnancy or lactation has not been established, don’t use during

pregnancy or lactation without professional advice.

SEKEM RENAL Composition:1- Licorice root:Liquorice root consists of the dried unpeeled or peeled whole or cut root and stolons of Glycyrrhiza glabra L., It contains not less than 4.0 per cent of glycyrrhizic acid

(C42H62O16, Mr 823).

Family: FabaceaeUSE:1-used as an adjuvant agent to improve the

therapeutic effects of certain drugs.

2-used to treat sore throats, mouth ulcers, stomach ulcers, inflammatory stomach conditions and indigestion.

3-used to combat food poisoning in modern Chinese herbalism.

Known hazards:It has been reported that excessive liquorice consumption can lead to cardiac dysfunction and severe hypertension.

1-Structural standards:1-Microscopic characters:

2-Organoleptic properties:

Odour: slight and characteristicTaste: very sweetColor: Unpeeled – yellowish or purplish brown to dark brown externally and yellowish internally.Peeled – pale yellow

3-Numerical values:173,176,184,219,233,278,279,337

,365,404,405,409,412,429,509,

669,671,672,673,679,683.

2-Pharmacobial standards:

1-Ash values:

Total ash:Not more than 7% (6, 7)

Acid-insoluble ash:Not more than 2% (1–3, 6, 7

Sulfated ash:Not more than 10% (2)

2-Extractive values:

Water-soluble extractive:Not less than 20% (8)

Dilute alcohol-soluble extractive:Not less than 25%

3-Moisture content:

During harvesting,roots contain 50% to 60%moisture.Roots are dried in sun for 2-3 days,and in shed for 10-12 days so that moisture content in roots,reduced by10%.

3-Analytical methods:1-chemical methods:

Assay for glycyrrhizin (glycyrrhizic acid, glycyrrhizinic acid) content (at least 4%) by means of spectrophotometric, thin-layer chromatographic or high-performance liquid chromatographic methods.

2-UV Spectroscopy:Glycyrrhizin: The absorption peaks are both at approximately 254 nm. (spectra A and B)Isoliquiritin apioside (7), Isoliquiritin (8), Isoliquiritigenin (12), spectra C; 7, 8, and 12 spectra D).

Chromatographic Methods:

1-High Performance Liquid Chromatographic Identification:

Sample Preparations: Extract 1.0 g of coarsely powdered Glycyrrhiza root in 50 mL of water by boiling for about 5 minutes, and filter. Repeat for 4-5 times or until the extract is colorless.Combine the extracts, concentrate to about 100 mL, and cool to room temperature.

Before injection, filter through a membrane filter of 0.45-um or finer pore size, discarding the first 5 mL of the filtrate.

Column:C18, 25-cm x 4.6 mm, 5-um

Mobile Phase: 0.14 g of anhydrous potassium dihydrogen phosphate in 900 mL of water, add 0.5 mL phosphoric acid, mix, complete to volume with water, and mix (Solution A); and acetonitrile (Solution B))

Elution: Gradient program

Flow rate: 1.5 mL/min

Detection: UV, 254 nm

Injection volume: 20 uL

4-Biological evaluation:

1- Microbiology:

The test for Salmonella spp. in Radix Glycyrrhizae products should be negative(B).

The maximum acceptable limits of other microorganisms are as follows:

For preparation of decoction:

aerobic bacteria-not more than 107/g

fungi-not more than 105/g

Escherichia coli-not more than 102/g.

2-Pesticide residues:

Normally, the maximum residue limit of aldrin and dieldrin for Radix Glycyrrhizae is not more than 0.05 mg/kg .

3-Heavy metals:

Recommended lead and cadmium levels are no more than 10 and 0.3 mg/kg, respectively, in the final dosage form of the plant material.

2- Chicory leaves:

Botanical: Cichorium intybus (LINN)

Family: Compositae / Asteraceae

Conservation status:Common and widespread; not of

conservation concern.

Medicinal Action and Uses:Chicory has properties similar to those of Dandelion, its action being tonic, laxative

and diuretic.

Application: It is used alone or in combination with other plant extracts in production of food or feed

additives.

Constituents:

A special bitter principle, not named, inulin and sugar.

Chicoric acid has been identified as the major compound in methanolic extracts of chicory.

Aliphatic compounds and their derivatives comprise the main fraction while terpenoids comprise minor constituents of the plant.

1-Structural standards:

1-Microscopic characters:

1- Large tracheids observed at 400x with Acidified Chloral Hydrate Glycerol Solution -

Alkemist Laboratories

2- An irregular mass of inulin observed at 400x with Acidified Chloral Hydrate Glycerol Solution - Alkemist Laboratories

3-Organoleptic properties:

Taste: The whole plant has a bitter taste, without acrimony or any very peculiar flavor. The taste is strongest in the root and weakest in the flowers.

Odor: specific

Colour: Green brown to brown amorphous powder which is slightly soluble in water and in ethanol 50%.

4- Testing for purity:

pH (10% solution): (4.0 to 7.0)

Level of protein: (14.70%)

Ash: (10.91%)

Crude ether extract: (3.68%)

crude fiber: (16.78%)

low concentration of total carbohydrates : (68.50%)

Sulphated ash, %, not more than: 10.0

Heavy metals, %, not more than: 0.001

Loss on drying, %, not more than: 5.0

Extractive values:

5-Analytical methods:1-Analysis of the extracts for coumarin by HPLC:

Standard solutions:

Coumarin(C9H6O2) (100mg) was dissolved in 40ml of acetonitrile and 60ml of water in volumetric flask. The final solution contained 100μg/ml of coumarin. For the working standard, 80μl of the stock solution was taken and made up to 1ml with 40:60 acetonitile and water respectively.

The chromatographic conditions for the analysis were as follows:

Mobile Phase: acetonitrile: water (40:60)

Separations: done in the binary mode using acetonitrile: water (40:60; v/v).

Flow rate: 1 ml min-1

Injection volume (“loop”): 20μl

UV detection: 274nm

Peak got at retention time:8.4min.

Peppermint leaves:

Peppermint is generally regarded as 'the world's oldest medicine', with archeological evidence placing its use at least as far back as ten thousand years ago.

Definition:Folium Menthae Piperitae consists of the dried leaves of Menth piperita.

Family:(Lamiaceae)

Selected vernacular names:pepper mint, balm mint, peppermint, piperita.

USE:Peppermint leaf is used for spastic complaints of the GI tract and gallbladder.It is used to aid the various processes of digestion: to combat gas, increase bile flow, heal the stomach and liver, etc.

The oil also contains menthone and menthyl esters.Other health benefits are attributed to the high manganese, vitamin C and vitamin A content; as well as trace amounts of various other nutrients such as fibre, iron, calcium, folate, potassium, tryptophan, magnesium, omega-3 fatty acids riboflavin, and copper.

Microscopic characteristics:It exhibits non-glandular and glandular trichomes as outgrowths; non-glandular trichomes uniseriate, papillose, 1-8-celled; glandular trichomes have 1-2-celled stalk and 1-8-celled glandular head containing the essential oil.

Upper epidermis composed of large, clear epidermal cells with sinuous, vertical walls and possessing few or no stomata.

palisade parenchyma, comprising a layer of columnar cells rich in chloroplasts; spongy parenchyma, of 4-6 layers of irregularly shaped chloroplastid.

3- Organoleptic properties:

Odor: characteristic, penetrating

Taste: characteristic, aromatic

Color: Brownish-green

4-Purity tests:

1-Foreign organic matter:

Not more than 5% stems, the diameter of which must be not more than 1.5 mm.

not more than 8% leaves showing brown stains due to Puccinia menthae.

not more than 2% other foreign matter.

Total ash:Not more than 15% according to the European pharmacopoeia (2); not more than 12% according to the African pharmacopoeia.

Acid-insoluble ash:Not more than 1.5%

Pesticide residues:The recommended maximum limit of aldrin and dieldrin is not more than 0.05 mg/kg.

Chemical assays:

Whole and cut leaves contain not less than 1.2% and 0.9% (v/w) essential oil, respectively, determined as described in the European pharmacopoeia.

Major chemical constituents:

The major constituent of the leaves is the essential oil (0.5-4%), which contains menthol (30-55%) and menthone (14-32%).

Menthol occurs mostly in the free alcohol form, with small quantities as the acetate (3-5%) and valerate esters.

5-Analytical methods:

1-Analysis by HPLC:

Ethanol extract of mint leaf TLC analysis of the ethanol extract exhibited 5 spots and UV-Vis spectrophotometry revealed 5 peaks.

However, carvone peak was not seen.

The extract was separated using column chromatograpy using a mixture of hexane and acetone (9.6:0.4).

The eluent had one compound which did not show absorbance under UV. This compound was solid, colourless and had a sweet odor.

According to literature, mint leaves have 76% menthol which is solid in nature, indicating that this compound could be menthol.

The IR spectrum revealed sharp bands at 3580 cm-1 to 3650 cm-1 due to the presence of phenol or alcohol group. The band at 1126.47 cm-1 was due to -C-C stretching.

The band at 2931.90 cm-1 was due to -C-H stretching. The band at 1259.96 cm-1 was due to-C-C- stretching.

GC-MS analysis revealed a sharp peak with a retention time of 13.48 minutes, with m/z value of 156.2, which corresponds to the molecular weight of menthol (155.6).

GCMS data and other spectral information confirm the isolated compound to be menthol.

Ammi vissnaga

Definition:Ammi Visnagae consists of the dried ripe fruits.Family: (Apiaceae)

Selected vernacular names:Spanish carrot, kella.

Geographical distribution:Indigenous to the Mediterranean region. Cultivated in North America and in Argentina, Chile, Egypt, India.

Use:In Egypt, a tea made from the fruit of this species has been used as an herbal remedyfor kidney stones. Laboratory rat studies show that the extract slows the buildup of calcium oxalate crystals in the kidneys and acts as a diuretic.

Mechanism of action:

Kidney stone patients are often given aqueous extracts of (Khella)in many middle and near eastern countries.

The mode of action of Khella as a kidney stone therapy is not well understood.

We postulated that the extract may inhibit crystallization of (CaOx) and prevent crystal retention within the kidneys.

Contraindications:

Ammi Visnagae is used in traditional systems of medicine as an emmenagogue, and its safety during pregnancy has not been established.

Therefore, in accordance with standard medical practice, the fruits should not be used during pregnancy.

Chemical constituents:

Khellin, a chemical compound obtained from A. visnaga, was used at one time as a smooth muscle relaxant, but its use is limited due to adverse side effects.

Amiodarone and cromoglycate are synthetic derivatives of khellin with fewer side effects which were developed for use in modern medicine.

Visnagin is another chemical compound which is found in A. visnaga.

1-Microscopic characters:

Powder characterized by fragments of pericarp with vittae, reticulate cells, vessels and fibres.

fragments with inner porous mesocarp cells crossed by oriented groups of endocarpal cells; and numerous fragments of endosperm.

Other fragments show cells of the brown seed coat and aleurone grains 4–10 μm in diameter,containing microrosette crystals of calcium oxalate2–5 μm in diameter.

Hairs and starch grains are absent.

2-purity tests:

Foreign organic matter:Not more than 2%

Total ash:Not more than 8%

Acid-insoluble ash:Not more than 3.5%

Loss on drying:Not more than 10%

Fruit of Ammi majus should be absent.• DIFFERENTIATING AMMI VISNAGA FROM AMMI

MAJUS:

Boil about 50 mg of Ammi visnaga Fruit, finely powdered, with 5 mL of water for 1 min and filter.

Add 1 or 2 drops of this decoction to 1 mL of sodium hydroxide solution (1 N in water).

Acceptance criteria: Rose-red color is obtained within 2 min (distinction from Ammi majusFruit, no rose-red color is formed).

Absence of starch:

Examine powdered Ammi visnaga Fruit under a microscope using water as a mounting medium.Add a few drops of iodine and potassium iodide TS1.

Acceptance criteria: No blue color is

observed.

Pesticide residues:

The recommended maximum limit of aldrin and dieldrin is not more than0.05 mg/kg .

Chemical assays:

Contains not less than 1% γ-pyrones(furanochromone derivatives) calculated as khellin, determined by spectrophotometry.

• MICROBIAL ENUMERATION TESTS <61>:

The total aerobic bacterial count does not exceed 105 cfu/g.

the total combined molds and yeasts count does not exceed 103 cfu/g.

the bile-tolerant Gram-negative bacteria does not exceed 103 cfu/g.

Absence of Salmonella species and Escherichia coli.

Successive extractives%:

Petroleum ether: 3.40

Chloroform (60-800C): 6.10

Absolute ethanol: 11.10%

ethanolic water extract: 19.50

pH values:pH value of 1% solution: 5.92

pH value of 10% solution: 5.64

Biological evaluation:

Phytochemical Screening and In-vitro Evaluation of Antioxidant and Antimicrobial Activities of the Entire Khella Plant (Ammi visnaga.L.).

5-Analytical methods:1-Analysis of the extracts by HPLC:

Tea bags including fruits of Ammi visnaga L. are used in Egypt as remedy for the treatment of kidney stones. .

HPLC for quantitative estimation of khellol glucoside (KG), khellin (KH) and visnagin (VS) simultaneously.

This occurred without any interference with other components despite the complex nature of some products especially the tea bags.

Perfect resolution between KG, KH and VS was possible through using a mobile phase consisting of:

water :methanol : tetrahydrofuran (50:45:5, v/v/v).Peaks were detected at245 nm.

The average retention time for KG, KH and VS was found to be 3.16 ^ 0.03, 7.60 ^ 0.04

and 8.90 ^ 0.03 min, respectively, for 10 replicates.

Chromatographic conditions:

The isocratic reversed-phase HPLC separation and quantitation were performed on a 250£ 4.6mm, 5 mm C18 column (Luna, Phenomenex, Torrance, CA, USA).

mobile phase at flow rate 1.3 mL/min.

Quantitation was achieved with UV detection at 245 nm based on peak area.

All determinations were performed at ambient temperature.

The injection volume was 20 mL.

The samples were filtered through 0.45-

mm pore size disposable filters.

HPLC chromatograms (20-mL injection volume) of laboratory-prepared

mixtures of 20mgmL21of KG, KH and VS.

Source:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3860133/

http://www.tloafghanistan.org/Liquorice%20Market%20Report.pdfhttp://www.botanicalauthentication.org/index.php/Glycyrrhiza_glabra_(root)

http://www.webmd.com/vitamins-supplements/ingredientmono-881-licorice.aspx?activeingredientid=881https://books.google.com.eg/books?id=gVDYAQAAQBAJ&pg=PT721&lpg=PT721&dq=Numerical+value+of+liquor

ice+root&source=bl&ots=Mvinys7Rqk&sig=uVCep-HHQL7WQiYir-RnC95URKE&hl=en&sa=X&ved=0ahUKEwiWzpCQz4jQAhUDfhoKHdfvDZMQ6AEIOjAF#v=onepage&q=Numer

ical%20value%20of%20liquorice%20root&f=falsehttps://books.google.com.eg/books?id=k4FHtNhrLyoC&pg=PA157&lpg=PA157&dq=Moisture+content+of+liquori

ce+root&source=bl&ots=Co7YJuxTUG&sig=7zlq4bASCNEtnJVV-XHmNOS7nNs&hl=en&sa=X&ved=0ahUKEwidkc3W4ojQAhVGnBoKHWETCPEQ6AEIVDAJ#v=onepage&q=Moi

sture%20content%20of%20liquorice%20root&f=falsehttp://www.kew.org/science-conservation/plants-fungi/glycyrrhiza-glabra-liquorice

https://www.hindawi.com/journals/isrn/2013/786151/

http://www.kew.org/science-conservation/plants-fungi/cichorium-intybus-chicoryhttp://www.vemo-vsv.com/en/products/herbal-extracts/cichorium-intybus/

http://www.vemo-vsv.com/en/products/herbal-extracts/cichorium-intybus/

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Dr/Nada Mansey