Juglans – Walnut leaf (Juglandis folium)
|Latin name of the genus:||Juglans|
|Latin name of herbal substance:||Juglandis folium|
|Botanical name of plant:||Juglans regia l.|
|English common name of herbal substance:||Walnut leaf|
Latin name of the genus: Juglans
Latin name of herbal substance: Juglandis folium
Botanical name of plant: Juglans regia L.
English common name of herbal substance: Walnut leaf
- 1. Introduction
- 2. Historical data on medicinal use
- 3. Non-Clinical Data
- 3.1. Overview of available pharmacological data regarding the herbal substance(s), herbal preparation(s) and relevant constituents thereof
- 3.2. Overview of available pharmacokinetic data regarding the herbal substance(s), herbal preparation(s) and relevant constituents thereof
- 3.3. Overview of available toxicological data regarding the herbal substance(s)/herbal preparation(s) and constituents thereof
- 3.4. Overall conclusions on non-clinical data
- 4. Clinical Data
- 4.1. Clinical Pharmacology
- 4.1.1. Overview of pharmacodynamic data regarding the herbal substance(s)/preparation(s) including data on relevant constituents
- 4.1.2. Overview of pharmacokinetic data regarding the herbal substance(s)/preparation(s) including data on relevant constituents
- 4.2. Clinical Efficacy
- 4.2.1. Dose response studies
- 4.2.2. Clinical studies (case studies and clinical trials)
- 4.2.3. Clinical studies in special populations (e.g. elderly and children)
- 4.3. Overall conclusions on clinical pharmacology and efficacy
- 5. Clinical Safety/Pharmacovigilance
- 6. Overall conclusions
- List of references
The herbal substance of European walnut (Juglans regia L.) leaves is mentioned in several
Ożarowski (1976), Miller and Murray (1998), Blumenthal (2000), Wyk and Wink 2004, Martindale
(2009), Wichtl (2004; 2009, PDR for Herbal Medicines (Gruenwald et al.(ed) 2000; 2004), German Comission E Monograph, Gehrman et al. (2005) and Natural Medicines Comprehensive Database (2009).
The herbal substance is described as the dried entire – margined leaflets freed from the rachis with faintly aromatic odour and astringent, somewhat bitter and harsh taste (Wichtl, 2004). By long preservation the leaves become brown and lose their aroma. Juglans regia leaves contain approximately 10% tannins (elegitannins) while juglone is unstable and in dry leaves it is present only in traces.
European walnut belongs to the family of Juglandacae which contains about 50 species in 8 genera. The deciduous tree native in
Paleobotanical data testify for a vegetation history of Juglans regia in southern Europe, Syria, China, Himalaya and in Central Asia (Kyrgyzstan) of the last 6800 years, where walnut possibly outlived the last glaciation (Beer et al. 2008). Climate analysis shows that the spread of walnut is favourably correlated with climate warming (Loacker et al. 2007).
Interestingly, the discovery of walnut shell used as necklace is described by Klichowska (1971) from archeological excavations of a cemetery of Roman influence period in Pruszcz Gdański.
Recorded paleobotanical data with reconstruction of vegetational history confirmed cultivation of Juglans regia throughout the past 6300 years in northeastern region of Italy (Lago della Costa) (Kaltenrieder et al. 2010).
In Mediterranean countries the use of Juglans regia leaves for medical purposes is a common tradition.
1.1. Description of the herbal substance(s), herbal preparation(s) or combinations thereof
• Herbal substance(s)
Leaves cut and dried
The leaves contain approximately 10% tannins of the ellagitannins type (Blumenthal 2000). According to DAC not less than 2%, calculated as pyrogallol (Wichtl, 2004).
This glucosidic form is decomposing by
The leaves contain about 3.4% of flavonoids (Wichtl 2004, Carnat et al. 1993), especially quercetin, hyperoside=quercetin
Several flavonoids (quercetin
Phenolic acids/phenolic composition
Previous data revealed the presence of phenolic acids:
Additionally the presence of neochlorogenic and
The total phenolic content in one of the above cultivars measured in methanolic and petroleum ether extracts were 94.39±5.63 and 92±1.40 mg GAE/g, respectively (Carvalho et al. 2010).
The leaf contains trace amounts of essential oil (obtained by the Tenax trapping method) of about
4 ppm, with monoterpene and sesquiterpene hydrocarbons. Monoterpenes are represented by:
Nahrstedt et al. (1981) received 26 terpenoid substances from steam distilled leaves of European walnut. The substances identified were: 21 monoterpenes, two sesquiterpenes, one diterpene, two compounds of terpenoid origin and eugenol. The steam distilled yield comprehends more sesquiterpenes and substances derived of esters of fatty acids, with the main substance –
Liu et al. (2004) performed an analysis of an essential oil extracted from Juglans regia leaves by hydrodistillation. By use of
Farag (2008) described detailed results of volatile compounds in the leaves from selected plants of the Juglandaceae family. In Juglans regia, using
The leaf also contains: cyclitols (11.2%), mucilage (7.6%), calcium (1.9%), potassium (1.4%) and 8.7% of ash (in brackets are average levels of 15 samples of dry leaves) (Carnat et al 1993).
The surface waxes from the leaves of Juglans regia exhibited presence of primary alcohols (41.6%), hydrocarbons (3%), esters (3.5%), aldehydes (5.5%) and fatty acids (8.4%) (Prasad & Gülz 1990).
From the fresh leaves progesterone was obtained with a yield of 0.00010% w/w (Pauli et al.2010).
Comminuted herbal substance (10% tannins)
•Combinations of herbal substance(s) and/or herbal preparation(s) including a description of vitamin(s) and/or mineral(s) as ingredients of traditional combination herbal medicinal products assessed, where applicable.
The herbal substance is also available in combination products. Main plants used in combination are:
Agropyron repens, Foeniculum vulgare, Foeniculum vulgare ssp. dulce, Hyssoppus officinalis, Juniperus communis, Mentha piperita, Phaseolus vulgaris, Plantago lanceolata, Potentilla anserina, Quercus robur, Rhamnus frangula, Sambucus nigra, Taraxacum officinale, Thymus vulgaris.
This assessment refers only to the use of Juglans regia leaves as single substance.
1.2. Information about products on the market in the Member States
Regulatory status overview
MA: Marketing Authorisation TRAD: Traditional Use Registration
Other TRAD: Other national Traditional systems of registration Other: If known, it should be specified or otherwise add ’Not Known’
This regulatory overview is not legally binding and does not necessarily reflect the legal status of the products in the MSs concerned.
Table 1. Products on the market
*Additional information for the WEU herbal tea from Juglans regia folium authorised in Germany: All preparations for which marketing authorisations for traditional use have been granted (with reference to former national regulations) are mentioned, regardless of the fact that some of them are not in accordance with current community law (as defined in directive 2004/24/EC). Traditional preparations were authorised in
**Additional comments: The herbal substance is not available in combination products.
Information on other products
The herbal substance is only available in combination products: >5
Main plants used in combination are: Agropyron repens, Foeniculum vulgare, Foeniculum vulgare subsp. dulce, Hyssoppus officinalis, Juniperus communis, Mentha piperita, Phaseolus vulgaris, Plantago lanceolata, Potentilla anserina, Quercus robur, Rhamnus frangula, Sambucus nigra, Taraxacum officinale, Thymus vulgaris.
Posology For topical use: apply on affected region at least in morning and in evening, but not more than 5 times daily.
For oral use: one level tablespoon (approx. 3.5 g) of tea mixture to one cup (0.2 litres) of hot water. Drink 1 – 3 cups of lukewarm tea daily.
Herbal tea orally in predisposition for diabetes and against obesity,
Topically against dermatomycosis.
Preparations (kind of extract, extraction solvent, DER)
1.Tincture (1:10.8 – 11.8) 19.03 g/20 ml (1.5 g Plantaginis lanceolatae folium, 1.5 g Anserinae herba, 1 g Hyssopi herba, 1 g Thymi herba, 1 g Quercus cortex pulv., 0.68 g Juglandis folium/ 100 ml of tincture) (Since 1997)
2.Herbal tea mixture: (1.05 g Phaseoli legumen, 0.52 g Taraxaci radix, 0.52 g Maydis stigma, 0.35 g Juniperi
3.Herbal tea mixture: (0.6 g Sambuci flos, 0.84 g Foeniculi dulcis fructus, 0.65 Graminis rhizoma, 0.42 g Juglandis folium, 0.42 g Menthae piperitae folium, 0.21 g Juniperi
4.Herbal tea mixture in filters: 0.25 g Sambuci flos, 0.23 g Foeniculi fructus, 0.19 g Graminis rhizoma, 0.12 g Juglandis folium/ filter [1 g]) (Since 1999).
The herbal substance is present only in multicomponent teas (containing >6 herbal substances each, teas were authorised in 1961 – 1963 and should be reoriented under the TU scheme)
1.3. Search and assessment methodology
Databases assessed up to March 2011:
Science Direct, PubMed, Embase, Medline, Academic Search Complete, Toxnet
Search terms: Juglans regia leaves, European walnut, juglone
2. Historical data on medicinal use
2.1. Information on period of medicinal use in the Community
According to ethnobotanical data, the decoction of Juglans regia leaves is still commonly used in traditional folk medicine of the Iberian Peninsula as a mouthwash, as dental antiseptic and against pharyngitis. Oral administration is used against renal stones and obesity (Agelet and Valles 2001; Blanco et al. 1999; Bonet et al. 1999).
In Bulgaria and Italy, walnut leaves are traditionally used as astringent and
The leaves of Juglans regia containing repellent and
In Sardinia Juglans regia leaves infusions are used in helminthiasis (Bruni et al. 1997).
In Turkey walnut leaves are frequently used externally for fever in sun stroke, in acne, and as an antifungal agent or to alleviate rheumatic pain (Erdemoglu et al. 2003; Kültür 2007).
Traditional use of Juglans regia as anthelmintic remedy is reported not only in Europe, but also in other parts of the world, such as the
In Lebanon, Juglans regia leaves are traditionally used as a remedy in rheumatic and neuralgic complaints mainly as compresses and cataplasms (El Beyrouthy et al. 2008).
In northern and in
In summary, the walnut tree has thousands of years of traditional medicinal use, to treat a wide range of complaints (Bruneton, 1993). Its use has been mentioned since ancient times. During the Middle Ages walnut preparations were used as a remedy for various skin ailments and were mentioned by Lonicerus (1564) and Matthiolus (1501 – 1577). Its therapeutic activity is presented by Schimpfky (1900) and in Madaus “Lehrbuch der Biologischen Heilmittel” (1938). The traditional use of Juglans regia in different diseases has been thoroughly documented in several handbooks and in folk tradition.
The leaves are contemporarily used to target skin ailments as an
Weiss and Fintelman 2000; Wichtl 2004, Wyk and Wink 2004, Frohne 2006).
2.2. Information on traditional/current indications and specified substances/preparations
Information on indications from products on the market
See section 1.2.
Juglandis folium (herbal substance):
Traditionally used in mild, superficial inflammation of the mucosa and skin and excessive perspiration of hands and feet as astringent and depurative.
Information on traditional indications from literature
Commission E Monograph. Juglans regia L., (Bundesanzeiger No 101, published June 1, 1990): Mild, superficial inflammations of the skin; excessive perspiration, e.g. of the hands and feet.
Herbal Drugs and Phytopharmaceuticals (Wichtl, 2004):
Topical route: In acne, eczema, scrophula, pyodermia, ulcers, excessive perspiration in the feet and hands.
Oral route: In folk tradition in gastrointestinal catarrhs, anthelmintic and as a “blood purifying remedy”.
Herbal Medicinals, Miller & Murray (1998): Atopic dermatitis (exudative phase)
Herbal Medicine, (Weiss & Fintelman, 2000): Dermatitis
Lehrbuch der Biologischen Heilmittel (Madaus, 1938):
Scrofulosis, hyperhidrosis, acne, herpes, crusta lactea, exanthema, eczema, tinea capitis.
Medicinal Herbs: A Compendium (Gehrman et al. 2005):
In mild, superficial skin inflammations; excessive perspiration, for example, of hands and feet.
Medicinal Plants of the World (Wyk & Wink, 2004):
Skin ailments: acne, eczema, fungal infections, inflammations, sunburn, perspiration, itchy scalp and ulcers. Decoctions are traditionally used against bedbugs and lice.
PDR for Herbal Medicines, Gruenwald et al.(ed.) (2000):
Mild, superficial inflammation of the skin, excessive perspiration.
Ziołolecznictwo, Ożarowski, (1976):
Mouthwash in stomatitis and inflammatory conditions of the throat, acne, neurodermatitis, eczema, hyperhidrosis.
On the basis of the information on traditional and current indications and the requirements for specified conditions of use to ensure a safe use, the following therapeutic indications are recommended for Juglans regia leaves to be included in the monograph:
Indication 1) Traditional herbal medicinal product for the relief of minor inflammatory conditions of the skin.
Indication 2) Traditional herbal medicinal product used in excessive perspiration of hands and feet.
2.3. Specified strength/posology/route of administration/duration of use for relevant preparations and indications
Information on posology, route of administration, duration of use from products on the market:
See section 1.2
Juglans regia leaves (comminuted herbal substance): 4 – 6 g as a decoction in 200 ml of boiling water. Apply as a dressing to the affected areas of the skin three times daily. Not to be used for more than
Information on posology, method of administration, duration of use from literature for relevant preparations
Commission E Monograph. Juglans regia L. (Bundesanzeiger No 101, 1990)
Dosage: Unless otherwise prescribed: Topically for cataplasms and partial baths: 2 – 3 g dried leaf per 100 ml water or equivalent preparations.
Duration of use: no information
Dosage: 2 – 3 g of the leaves / 100 ml of boiling water, heat for 15 min and then strain. Topical application for cataplasms or rinses
Herbal Drugs and Phytopharmaceuticals (Wichtl, 2004)
Dosage: Place 1.5 g of finely cut dried leaf into cold water. Heat to boil and then strain after 3 – 5 min. Oral route: drink one cup of tea 1 – 3 times daily.
Topical route: For cataplasms and rinses, decoct 5 g of dried leaf in 200 ml water. (1 teaspoon=about 0.9 g).
Duration of use: no information
Herbal Medicinals (Miller & Murray, 1998)
Dosage: Topically. Place 5 g (5 teaspoons) of the chopped drug in 200 ml water.
Duration of use: no information
Herbal Medicine (Weiss & Fintelman, 2000)
Oral route: add one tablespoonful of the finely chopped drug to ½ liter cold water, bring to a boil and
strain after three to five minutes. Drink two to three cups each day.
Topical route: The decoction for compresses is made using around five tea spoonfuls of the drug. Duration of use: no information
Lehrbuch der Biologischen Heilmittel (Madaus, 1938)
Oral route: 2 – 3 spoonfuls (=3 – 4.5 g) of leaves in hot water (cup). Drink up to four times daily. Bath: 0.5 – 1.0 kg of leaves, boil in a few liters of water and add to the morning and evening bath. Maximum dose is not described.
Duration of use: no information
Medicinal Herbs: A Compendium (Gehrman et al. 2005)
Dosage: 2 – 3 g (2 – 3 teaspoons)/150 ml, cold maceration, heat to boiling point for 15 min, for poultice and as bath additive
Duration of use: no information
Medicinal Plants of the World, Wyk and Wink, (2004)
Dosage: An infusion is made by heating 1.5 g of the herb in a cup of water to boiling point. For dressings or lotions, a decoction of 5 g in 200 ml water is recommended.
PDR for Herbal Medicines, Gruenwald et al. (ed.) (2000)
Dosage: Comminuted drug for decoctions for external use – soak 2 teaspoonful of drug in 1 cup of water, boil and strain.
An infusion is prepared by using 1.5 g of finely cut drug, soaked in cold water, brought to simmer and strained after 3 to 5 minutes.
The average daily dose for external use is 3 to 6 g of drug.
Duration of use: no information.
Ziołolecznictwo, Ożarowski, (1976)
Dosage: Place 20 – 30 g of the leaves in 500 ml of hot water for mouthwash, compresses and cataplasms.
Duration of use: no information
On the basis of the information on traditional and current dosages and the requirements for specified strength and specified posology, the following is recommended for Juglans regia leaves to be included in the monograph:
Adults and elderly
Comminuted herbal substance as decoction preparation for cutaneous use: 4
The use in children and adolescents under 18 years of age is not recommended.
Duration of use
Not to be used for more than 1 week.
3.1. Overview of available pharmacological data regarding the herbal substance(s), herbal preparation(s) and relevant constituents thereof
In vitro experiments
Almeida et al. (2008) described scavenging effects of an ethanol:water (4:6) extract of Juglans regia leaves. Scavenging activity was established on reactive oxygen species (ROS) [hydroxyl radical (HO), superoxide radical (O2), peroxyl radical (ROO), hydrogen peroxide (H2O2)], reactive nitrogen species (RNS) [nitric oxide (NO), and peroxynitrite anion
Trolox equivalents/mg extract. The authors suggest that the observed antioxidant activities result partly from the presence of phenolic compounds in the extract (total phenolics content was 270 ± 3 mg of gallic acid equivalents/g of lyophilized extract).
Another study of radical scavenging activity of acetone, ethyl acetate and methanol extracts of several plants, Juglans regia leaves included was presented by Miliauskas et al. (2004). Dried material
(6 – 10 g±0.01 g) and 100 ml of solvent was used. The extracts were filtered and concentrated at 400C. Radical scavenging activity was established by use of a spectrophotometric method against the stable
Ethanolic extracts of several plants, Juglans regia leaves included, were tested for their antioxidant activity to inhibit the superoxide anion formation and lipid peroxidation levels in vitro (Çoban et al., 2003). The dried and chopped leaves of Juglans regia (20 g) were extracted with ethanol (75% aqueous, 300 ml) for 24 h using the Soxhlet apparatus. Inhibitory effects of the extract on superoxide formation and on lipid peroxidation are shown in the Table 1 and Table 2).
Table 1. Inhibitory effect of Juglans regia extract on superoxide anion formation (after Çoban et al., 2003).
Each value represents the mean±SD of
Ethyl alcohol only, control for the extract
Table 2. Inhibitory effect of Juglans regia extract on lipid peroxidation (after Çoban et al., 2003).
Each value represents the mean±SD of
Ethyl alcohol only, control for the extract
Antioxidant capacity of Juglans regia aqueous extract was established via the haemolysis inhibition of human erythrocytes and bacteriophage P22 and via the Salmonella typhimurium model protection from the oxidant challenge by H2O2 (Giao et al. 2010). To prepare the aqueous extract 1 g of finely milled powdered dried leaves were added to 110 ml of boiling water for 5 min and filtered through 0.45 μm filters and lyophilized. For both the haemolysis and the phage method extracts were reconstituted to obtain 0.0025% (w/v), or at ca. 0.02% (w/v). The assays showed positive antioxidant effects for the extract of Juglans regia leaves in the haemolysis test, but no effect in the bacteriophage test.
Antimicrobial activity and antifungal
Çitoğlu and Altanlar (2003) tested antibacterial and antifungal activity of several plants, Juglans regia included. The dried and chopped samples of 20 g of Juglans regia leaves were extracted with ethanol (75% aqueous, 150 ml) for 24 h by using a Soxhlet apparatus. The extract was tested using the disc diffusion method against
Candida krusei) for its antimicrobial activities. The antimicrobial effects were compared with the antibiotic ampicillin, and the antifungal agent fluconazole and positive control (ethanol) (Table 3).
Table 3. The inhibition zones diameter of the aqueous extract of Juglans regia. (after Çitoğlu and Altanlar, 2003).
Diameters of the inhibition zones (mm)
The results of the test show good antimicrobial activity of the aqueous Juglans regia extract.
Kong et al. (2008) reported, that juglone displays different inhibiting activity against 3 main enzymes from H pylori: cystathionine
The antibacterial activity of methanolic and aqueous Juglans regia extracts was described by Mehrabian et al. (2000) against Gram +, airborne Bacillus cereus and Bacillus mycoides. Fifty g of powdered plant was added to 100 ml of each solvent: water or methanol. After 24 h the extracts were purified by distillation. The plates with cultured bacteria were incubated at 37oC for 24 h and 1 ml of 1:10 or 1:20 dilution of the aqueous and methanolic extracts were added respectively do the cultured plates. Indication of the antibacterial activity was estimated after 48 – 72 h. The aqueous extract of of Juglans regia leaves induced growth inhibition of Bacillus cereus and Bacillus mycoides, (diameter of zone of inhibition ~ 15 – 20 mm), while the methanol extract inhibited the growth of Bacillus mycoides only (diameter of zone of inhibition ~ 20 mm).
Darmani et al. (2006) examined the activity of the aqueous extract of Juglans regia on proliferation of Balb/C 3T3 mouse fibroblasts and viability of cariogenic bacteria (Streptococcus mutans, Streptococcus salivarius, Lactobacillus casei and Actinomyces viscosus). The results showed the significant increase in cell proliferation by 255% (p<0.0001). The walnut extract exhibited antibacterial effect against cariogenic bacteria: where the most sensitive were: A. viscosus, followed by S. mutans, S. salivarius, whilst L. casei appeared as the most resistant.
Pereira et al. (2007) investigated leaves of 6 different cultivars of Juglans regia for its antioxidant and antimicrobial activity. After determination of the phenolic content (naphtoquinones and flavonoids), the antimicrobial activity was screened on Gram positive (Bacillus cereus, B. subtilis, Staphylococcus aureus) and Gram negative (Pseudomonas aeruginosa, Escherichia coli, Klebsiella penumoniae) bacteria and fungi (Candida albicans, Cryptococcus neoformans). Juglone was not found in the samples (due to low solubility in water and volatile in steam). Three powdered subsamples (~ 5 g) were extracted with 250 ml of boiling water for 45 min and filtered, frozen, lyophilized and redissolved in water at concentrations of 100 mg/ml and 10 mg/ml. All tested cultivars showed antimicrobial activity, Gram positive (Bacillus cereus) being the most susceptible (MIC 0.1 mg/ml). The walnut leaves of the Lara cultivar were most active, therefore its extract was tested against 18 Staphylococcus sp. strains isolated from sputum, pus and blood. The most susceptible were the strains isolated from sputum (MIC’s range of 0.1 mg/ml), while MIC’s for the strains isolated from pus and blood ranged between 0.1 and 1 mg/ml. The Lara cultivar exhibited also the highest antioxidant activity in three other tests: reducing the power assay (EC50=192 mg/ml), scavenging effects on
Qadan et al. (2005) tested antimicrobial activity of a Juglans regia extract against acne developing bacteria: Propionibacterium acne, Staphylococcus aureus and Staphylococcus epidermidis. Walnut leaves (500 g) were air dried and extracted with acetone: H2O2 (7:3.5 l). The extracted material was evaporated to 1.5 l, filtered and defatted. Later it was shaken with ethyl acetate and the water residue (46 g) was subjected to column chromatography with
(10% w/v) at room temperature, the aqueous extract was obtained by decoction of 30 g of material (6% w/v) and then
Table 4. Antimycobacterial activity of Juglans regia extracts (after
All biological assays were done at least by duplicate
NA _ they did not present activity at 1000 μg/ml
In conclusion, the leaves extracts did not have evident antibacterial activity. As the MIC value for the methanol extract was ≥ 100 μg/ml, the extract was considered inactive against M. tuberculosis (Cos et al. 2006).
Antilisterial effects of Juglans regia were evaluated by the agar diffusion and the macrodilution method (Altanlar et al. 2006). The leaves of Juglans regia (20 g) were extracted with ethanol (75% aqueous, 150 ml) for 24 h by using Soxhlet apparatus.
The tests were performed against L. monocytogenes, L. ivanovii, L. murrayi and L. innocua. Minimum inhibitory concentrations (MIC) of the evaluated Juglans regia extract were 12.5 μg/ml against
L.monocytogenes and L. murrayi, 25 μg/ml against L. ivanovii and without any effect against
L. innocua. MIC’s of the active control ofloxacin were: 3.125 μg/ml against L. monocytogenes and
6.25μg/ml against other species.
The antifungal activity of dried aqueous extracts of 22 medicinal plants, Juglans regia leaves included (100:1) was tested against fungal isolates in culture: Microsporum canis (S14, S20, and SH41),
Trichophyton mentagrophytes (SH13, SH1, SH8), and Trichophyton violaceum (S5, SH32, SH38). Fungi were isolated from patients with Tinea capitis. As reference the antibiotic griseofulvin was used. Results are presented in Table 5
Table 5. Mycelial inhibition of tinea capitis dermatophytes (after
Means* of percentage mycelial inhibition of tinea capitis dermatophytes
*Means of three replicate plates for each of three isolates for each species.
**Concentrations of reference antibiotic (MIC) was 0.6 μg/ml for M. canis, 2.5 μg/ml for T. mentagrophytes, and 0.6 μg/ml for T. violaceum.
The MIC values of Juglans regia extract (15 μ/ml medium) showed high: 97.6 to 100% inhibition of mycelial growth and were comparable with griseofulvin activity. However, according to other experiments Juglans regia was inactive against Candida albicans.
Min et al. (2002) reported the antiviral activity of quinones, juglone included, on RNase H activity associated with
Mouhajir et al. (2001) investigated antiviral activity of methanol extracts of 75 Moroccan plants, Juglans regia included. The extracts were prepared with use of a 100 – 200 g of the dried plant material soaked in 100 – 200 ml of methanol, filtrated and washed again with another 200 ml aliquot of methanol. The filtrates were evaporated to dryness and finally diluted to a concentration of
2 mg/ml. Extracts were evaluated against: herpes simplex virus (HSV), Sindbis virus (SINV) and poliovirus (polio) at
Table 6. Antiviral activity of Juglans regia methanol extract (MIC μg/ml) (after Mouhajir et al. 2001).
Light and dark activities
In vitro experiments
Antioxidant and antiproliferative activities of the methanolic and petroleum ether extracts of Juglans regia leaves were described by Carvalho et al. (2010).
Antiproliferative activity (IC50, mg of methanolic extract/ml) of the Juglans regia leaves towards cultured
Enzyme activity inhibition
Muto et al. (1987) described the inhibition of microsomal cytochrome P450 monooxygenase activity in rabbit liver and in human placenta. Strong inhibition of synthesis of placental estrogen was seen. The authors explain the inhibition by direct interaction of the naphtoquinone (juglone) with the prosthetic heme group.
Significant inhibition of urease activity , the enzyme responsible for catalysing the hydrolysis of urea, due to juglone, has been found by Kot et al. (2010). The authors supposed that
Antityrosinase activity of methanol extracts of Castanea sativa stem bark, Eucalyptus camaldulensis leaves and Juglans regia leaves were tested by Özer et al. (2007). Tyrosinase inhibition plays an important role in the treatment of uneven skin hyperpigmentation. Plant material (100 g) was extracted with 80% methanol, filtrated and evaporated to dryness under vacuum. The quantity of ellagic acid in the Juglandis folium extract was the highest: 16.25±0.512% (n=6±SD). Walnut extract manifested as well the highest antityrosinase activity with IC50 = 505 μg/ml, which was found to be 1.2 and 1.8 fold higher than that of Eucalyptus camaldulensis leaves and Castanea sativa stem bark, respectively. The Author concluded that the extracts in gel formulations containing Juglans regia could be used as
In the study of Perusquia et al. (1995) the aqueous extract of Juglans regia leaves inhibited in a concentration dependent manner (0.5 – 12 mg/ml) the maximal contractile response induced by noradrenaline in isolated rat thoracic aorta.
In vivo experiments
Mohammadi et al (2011) reported from in a model of streptozocin diabetes in rats a significant antidiabetic activity of the aqueous extracts of Juglans regia leaves. The extracts were prepared from powdered dry leaves (700 g), which were subjected twice for 24 h to extraction with 90% ethanol in room temperature. The filtrate was evaporated until dry to 80.4 g of powder, which was later diluted in water to receive doses of 200 and 400 mg/kg. Diabetes was induced in rats by single injection of
55 mg/kg i.p. of streptozocin (STZ) and identified in animals with glucose level over 300 mg/dl. Four groups of 8 rats were used in the experiment. After ten days diabetic groups III and IV received orally 200 and 400 mg/kg of the extract of Juglans regia leaves for 28 days. The control (I) group received buffer i.p.. After autopsy, glycosylated hemoglobin, triglyceride, cholesterol, LDL, VLDL and HDL levels were determined (Table 7.)
Table 7. The effect of the extract of the Juglans regia leaves on glycosylated hemoglobin, triglyceride, cholesterol, LDL, VLDL and HDL levels (after Mohammadi et al. 2011).
The values are mean ± SE for 8 rats in each group. Means with different superscript (a, b, c, d) within a column are significantly different from each other at P<0.05 as determined by Duncan’s multiple range test.
The glucose concentration in blood of the II and IV group was significantly diminished. The extract of Juglans regia leaves at the dose of 400 mg/kg after 21 injections restored the glucose levels almost to the control values. Also insulin levels in group III and IV were enhanced and in group IV 9400 mg/kg) reached almost of those in the control (I) group.
Similar results were presented by Asgary et al. (2008) with an ethanolic extract of Juglans regia leaves in the
Similar antidiabetic action of Juglans regia leaves extracts were also described by Gholamreza and Hossein (2008) and Dzhafarova et al. (2009) in Wistar rats with experimental diabetes.
Inhibitory effects of juglone on
Insect growth regulatory activity
In order to develop alternative strategies for the protection of important crops, juglone was found to induce lethal effects on the growth of insect red cotton bug (Dysdercus koenigii Fabr.) in concentrations of 1 – 10 μg/nymph (Banerjee et al. 2001; Magdum et al. 2001).
Experiments in vitro were performed with the aqueous extract of several plants, Juglans regia included against larvae of Trichoferus griseus living on Pinus brutia L (Civelek and Colak 2008). Insecticidal effects of Juglans regia extract were compared with insecticide Bensultap and synthetic chemical preservation (chromated copper arsenate, Tanalith C). The extract was prepared from 2 g plant sample and extracted with 100 ml of water using a Soxhlet hot water extractor. Afterwards the extract was stored at 50C until use. The average number of alive larvae (± SE) at the end of the experiment were: 0.4±0.05 (p<0.05) after Juglans regia extract, 0.0±0 after Bensultap – a positive control, and 0.666±0.059p<0.05) after Tanalith C.
3.2. Overview of available pharmacokinetic data regarding the herbal substance(s), herbal preparation(s) and relevant constituents thereof
No data are available concerning Juglans regia leaves pharmacokinetics due to its complex phytochemical composition.
Table 8. Disposition of
(n=4) 24 or 72 h following administration. (after Chen et al. 2005)
18.2 ± 3.6
21.3 ± 1.6
23.6 ± 2.8
32.8 ± 2.5
2.5 ± 0.7
6.3 ± 0.6
63.2 ± 6.2
65.0 ± 3.6
69.2 ± 3.5
23.3 ± 2.0
1.3 ± 0.4
5.3 ± 0.4
1.1 ± 0.2
1.6 ± 0.1
0.7 ± 0.1
3.0 ± 0.9
28.6 ± 3.9
6.4 ± 1.9
4.8 ± 0.4
5.7 ± 1.6
0.1 ± 0.1
3.8 ± 0.9
3.6 ± 0.8
0.6 ± 0.1
1.2 ± 1.0
0.1 ± 0.1
0.1 ± 0.1
80.2 ± 6.5
38.7 ± 2.0
Skin at the
2.9 ± 0.4
8.4 ± 2.1
89.2 ± 2.1
93.6 ± 3.5
93.6 ± 2.1
88.4 ± 2.4
96.0 ± 8.4
67.2 ± 2.1
aFaecal contamination of the urine made the distribution between urine and faeces uncertain bNot determined
The study shows that less than 50% of an oral dose (0.1 to 10 mg/kg) and less than 20% of a topical application on skin (4 mg/kg) were absorbed. After absorption, juglone radioactivity was almost completely excreted in faeces and urine in 24 h after oral administration, with only 1 – 3% left in the body. A comparatively high concentration of juglone was present in the kidneys, regardless of the route of application. After oral administration of juglone in rats, five metabolites were identified in the urine as a result of two electron reduction conjugated later by glucuronide and sulfate.
Overview of pharmacokinetics
The pharmacokinetics of
There is no data on juglone pharmacokinetics in humans.
Labelled [14C]hydroquinone pharmacokinetics were studied after topical application of a cream containing 2% hydroquinone to the skin of human volunteers (Wester et al., 1998). After 24 h a 45% bioavailability was found with a rapid and continuous movement of the hydroquinone to the stratum corneum. The maximal plasma concentrations were found at about 4 h after application and most radioactivity was eliminated with the urine within 24 h as glucuronides.
Due to lack of data on the pharmacokinetics of walnut leaves no conclusions can be drawn.
3.3. Overview of available toxicological data regarding the herbal substance(s)/herbal preparation(s) and constituents thereof
Walnut has been reported allelopathic, i.e. to actively limit the growth of various other plant species, including herbs, shrubs, deciduous trees nearby (von Kiparsky et al. 2007; Weir et al. 2004). In Turkey, the practice of intercropping with the use of Juglans regia is popular (Ercisli et al. 2005). It is reported, that wood shavings or heartwood of black walnut (Juglans nigra) causes laminitis in horses, but such effects have not been observed for Juglans regia (Harman 2002).
Juglone is toxic to mammalian cells in vitro and also to small animals in vivo (Soderquist 1973). Juglone is toxic to fish at <0.1 mg/l and can kill the fish before degrading in the natural environment (Marking 1970).
It is also very toxic to insects such as Drosophila larvae. In adult males which are exposed to a 10% aqueous sucrose solution of juglone a strong mutagenic response was found, mainly in first the generation. The size of the brood was significantly diminished (Clark 1982; Harada 1994)
Juglone has a potential for human exposure as a result of appearance in hair dye formulations and use in dietary supplements. However, very low concentrations of juglone present in the decoction do not pose a health hazard. To determine the amount of juglone in herbal preparations, the content in the decoction/infusion of Juglans regia leaves by GC/MS analysis was analysed by Matławska et al. (2012, submitted). The infusion contained detectable juglone amounts 0.1%, but the amount of juglone in the decoction was found below the detection limit (<0.0003%). Simulation of the conditions found in the lumen of the stomach and on the human skin surface at body temperature of 37°C, induced by the change of pH of the tested solutions to 2.0 or 4.0 values, does not increase the concentration of juglone (<0.0003%). This means that juglone is not present in the acidic environment of the skin and in the gastric juice after topical or even oral ingestion. The authors have demonstrated that the decoction of walnut leaves, is the most appropriate for safe use for topical application on to the skin, due to very low juglone content.
Acute toxicity of juglone was studied by Aithal et al (2011). It was found that oral LD50 for rat is 112 mg/kg, and for the mouse 2.5 mg/kg. Repeated dosing produced an accumulation of juglone
adducts in the kidney. After
An old tradition, of using ichthyotoxic plants in Europe is confirmed by ethnobotanical records as early as 1255 when in Spain such plants were formally made public and their use forbidden. In folk tradition Juglans regia is one of the 32 plants registered in common use for fish poisoning, but this kind of practice is currently curtailed (Alvarez Arias 2000).
Juglone acts as a generator of superoxide anion radicals. The cytotoxic activity of juglone depends on the decrease in glutathione due to enzymatic redox cycling of naphtoquinones and production of DNA and protein adducts as elucidated by Ross et al. (1986), Öllinger and Brunmark (1991) and O’Brien (1991). Similar results were obtained by Murakami et al. (2010), where juglone markedly stimulated the lipid peroxydation. Juglone also produced
Cytotoxicity of juglone
Juglone induced cell death by apoptosis and necrosis through diverse mechanisms such as induction of oxidative stress, cell membrane damage and clastogenic effects (Aithal et al. 2009; Babula et al. 2009; Bayer et al. 2005; Inbaraj and Chignell 2004; Öllinger and Brunmark 1991).
Cytotoxic activity of juglone was demonstrated after 72 h of incubation against leukemia
Control groups received DMSO only. The supernatant was replaced by fresh medium containing MTT (0.5 mg/ml) and after 3 h the MTT formazan product absorbance was measured. Doxorubicin (Dox) was used as positive control. The MTT analysis showed that the IC50 juglone values ranged from 7.6 to over 28.7 µM according to cell type after 72 h of incubation. IC50 ranged in
Table 9. Cytotoxic activity of juglone and its derivatives on tumor cell lines, on peripheral blood mononuclear cells (Montenegro et al. 2010)
*Data are presented as IC50 values and 95% of confidence interval for leukaemia
In another similar study cytotoxic and genotoxic effects of juglone were demonstrated against the melanoma B16F1 cell line.
Contrary effects were described by Monks et al. (1990) in the study of induction by quinones (juglone) epidermal ornithine decarboxylase (ODC) and promotion skin tumors growth in mice. Single topical application of juglone produced
It was found that juglone inhibits several enzymes’ activity, including the members of the parvulin PPIase family (Pin1) by modifying sulfhydryl groups (Hennig at al 1998; Maruyama and Furutani 2000). Pin1 plays an important role in oncogenesis and its expression is increased in many human cancers.
Juglone, independently of its Pin1 irreversible inhibition, can inhibit
Juglone acts as an active inhibitor of RNA processing by blocking transcription of RNA polymerase II (Chao et al. 2001). Moreover, juglone can induce apoptosis and/or necrosis probably by subsequent activation of caspases as reported previously for other naphtoquinones such as benzobijuglone from
Juglans mandshurica (Li et al. 2007).
Juglone treatment inhibited intracellular mammalian protein thioredoxin reductase (TrxR) causing formation
In colonies of human cancer cells, juglone induced apoptosis: it was found strongly cytotoxic and induced
In another model of cytotoxicity, juglone inhibited growth and induced apoptosis in human gastric cancer
The cytotoxic mechanism is induced by the activation of the mitochondrial death pathway with apoptosis and necrosis of gastric cancer cells (Table 10, Ji et al. 2009).
Table 10. Effect of juglone on the cell apoptosis of
(after Ji et al. 2009).
*Compared with control P<0.05. **Compared with control P<0.01.
HCPT (Hydroxycamptothecine) – active control
Genotoxicity of juglone and Juglans regia extracts
Juglone tested with S. typhimurium in the presence of
Tests with S. typhimurium without
A positive value of genotoxic activity of juglone in the two tests of w/w SMART assay of Drosophila melanogaster (498% of mosaic eyes induced per mM of compound at the lowest effective dose) was presented by Gaivao et al. (1999). The Ames test data for the other naphtoquinones were variable with some positive results.
In conclusion the genotoxicity of juglone is considered scarce and weak.
The evaluation of genotoxicity of the Juglans regia extracts showed significant damage in the comet assay in concentrations above 250 ppm but not in the VITOTOX test. The extracts in the Ames assay showed potent antimutagenic effects against the direct acting mutagens NPD, sodium azide, and the S-
No published data could be found on the genotoxicity of European walnut leaves.
3.4. Overall conclusions on
It can be concluded that despite that juglone as a quinone compound has been linked to mutagenic action, it is unstable and in dry leaves it is present only in traces. On the basis of existing findings and comparisons with other quinones, genotoxicity seems not conclusively evaluated and may be limited, weak and probably meaningless to humans following exposure to small amounts of juglone.
Experimental preclinical data show antioxidant, antiviral, antibacterial, antifungal, antidiabetic and
The published data on pharmacological activities support the traditional use of preparations containing Juglans regia in the proposed indications.
4. Clinical Data
4.1. Clinical Pharmacology
4.1.1. Overview of pharmacodynamic data regarding the herbal substance(s)/preparation(s) including data on relevant constituents
There are no data on human pharmacodynamics.
4.1.2. Overview of pharmacokinetic data regarding the herbal substance(s)/preparation(s) including data on relevant constituents
There are no data on human pharmacokinetics.
4.2. Clinical Efficacy
4.2.1. Dose response studies
There are no specific data available on
4.2.2. Clinical studies (case studies and clinical trials)
No studies were published on
4.2.3. Clinical studies in special populations (e.g. elderly and children)
No information available.
4.3. Overall conclusions on clinical pharmacology and efficacy
There are no data available from controlled clinical studies, therefore the medicinal use of Juglans regia leaves has to be regarded as traditional only.
5. Clinical Safety/Pharmacovigilance
There are no adverse effects reported from the Member States, however allergic reactions to Juglandacae family should be considered.
5.1. Overview of toxicological/safety data from clinical trials in humans
There are no data from clinical trials available.
5.2. Patient exposure
5.3. Adverse events and serious adverse events and deaths
Allergic reactions following contact with walnuts are common. Both, the tree and its fruit can be responsible. Moreover cases of allergic contact dermatitis to walnut wood have been described. The history of 14 month of contact dermatitis with hand eczema, were seen in a 35
The intense pigmentation and acute irritant contact dermatitis with large blisters involving the palms and fingers due to the contact with Juglans regia juice was described in Southern Italy (Bonamonte et al. 2001). After 5 days treatment with compresses of aluminium acetate solution the irritant contact dermatitis symptoms had declined, however pigmentation lasted 3 weeks.
Two cases of pigmentation and acute irritant contact dermatitis were described by Neri et al (2006) in children playing with green walnut husks.
Ethnic customs of using Juglans regia preparations as a tooth cleansing method, can induce pigmentation in the oral cavity (Ashri and Gazi 1990; Eisen 2000).
5.4. Laboratory findings
No data available.
5.5. Safety in special populations and situations
There are no reports on the use of Juglans regia leaves in children. The use of walnut leaves is therefore not recommended in children younger than 18 years of age.
None reported for Juglans regia leaves preparations. However, it is reported in literature that Juglans regia due to high tannin content may cause alkaloids to become insoluble and precipitate, as well as reduce the absorption of other drugs (Herr 2005).
Some authors mention that tannins may also interfere with iron absorption.
Use in pregnancy and lactation.
The walnut leaves bark should not be used during pregnancy and lactation.
Effect on ability to drive or operate machinery or impairment of mental ability
5.6. Overall conclusions on clinical safety
There are no reports of adverse effects of walnut leaves from Member States.
The allergic reactions in patients allergic to Juglandaceae should be considered.
6. Overall conclusions
The available data are sufficient to include the traditional use of specified preparations of Juglans regia leaves in a monograph of the European Community. European walnut leaves fulfil the requirement of safe therapeutic use for at least 30 years (15 years within the Community, Directive 2004/24/EC).
Indications for treatment: 1) Traditional herbal medicinal product for mild superficial inflammatory conditions of skin. 2) Traditional herbal medicinal product used in excessive perspiration of hands and feet.
Due to the lack of adequate data on genotoxicity a list entry for Juglans regia leaves cannot be recommended.
There are some concerns about serious side effects with Juglans regia leaves due to presence of juglone. However, juglone is unstable and in dry leaves it is present only in traces.
There are reported side effects concerning allergic reactions due to the Juglans regia leaves preparations used. No serious adverse events with a therapeutic posology of the herbal preparations are reported.
Despite the insufficiency of toxicological data, levels of exposure associated with the use of walnut leaves by topical route of administration for limited time most probably have no relevance to human health and do not result in significant risks.
It can be concluded that the benefit/risk assessment for Juglans regia leaves preparations is positive for use in therapeutical dosages in specific conditions of the mild superficial inflammatory conditions of skin and in excessive perspiration of hands and feet.