Walnut leaf – Juglandis folium (Juglans regia L.)

Latin name of the genus: Juglandis folium
Latin name of herbal substance: Juglans regia l.
Botanical name of plant: Herbalref.com
English common name of herbal substance: Walnut leaf

Latin name of the genus: Juglandis folium
Botanical name of plant: Juglans regia L.
English common name of herbal substance: Walnut leaf

Juglandis-folium - Walnut leaf at herbalref.com

Table of Contents

1. Introduction

The herbal substance of European walnut (Juglans regia L.) leaves is mentioned in several well-known handbooks and sources such as Madaus (1938), Deutschen Arzneibuch (1941), Wren (1975),

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 South-eastern Europe is now cultivated and distributed in North temperate and subtropical regions of Europe, Africa, Asia and America. It is native in a region stretching from the Balkans eastward to the Himalayas and southwest China. The largest forests are in Kyrgyzstan, where trees occur in extensive, nearly pure walnut forests at 1,000–2,000 m altitude. The tree is possibly native in Greece and elsewhere in the Balkan Peninsula; widely cultivated and locally naturalised in Europe (Flora Europea 2011). The Latin name of the plant Juglans originates from the Latin Jupiter and glans (acorn) meaning “Jupiter nuts”.

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).

Naphtalene derivatives

Naphthoquinones are oxygen-derivatives of naphthalene. The most known naphthoquinone constituent is juglone (5-hydroxy-1,4-naphtoquinone) which occurs in fresh plant (leaf, stain) as glycoside of

reduced form: 4β-D-glucoside of α-hydrojuglone=4β-D-glucoside of 1,4,5-trihydroxynaphthalene (2% in the stain, 0.6% in the leaves), but also in free state, particularly in the epicuticular leaf wax (to about 30%), (Wichtl 2004, Evans 2009, Hazra et al. 2004; Prasad and Gülz 1990).

This glucosidic form is decomposing by hydrojuglone-β-D-glucopyranoside-β-glucosidase to juglone (Morant 2008). Juglone is unstable and easily polymerized into yellow, brown and black pigments, so in the older leaves or dry leaves it is present only in trace amounts. When dried leaves were used for extract preparation, juglone was not detected, which means that this compound is not suitable for use in quality control of the dry plant (Amaral et al. 2004; Babula et al. 2009a, Gîrzu et al. 1998; Wichtl 2004; Wójcik 1984).


The leaves contain about 3.4% of flavonoids (Wichtl 2004, Carnat et al. 1993), especially quercetin, hyperoside=quercetin 3-O-galactoside (0.6% according to Carnat et al. 1993), and 0.2-0.6% of quercitrin=quercetin 3-O-rhamnoside (Wichtl 2004). Kaempferol and kaempferol 3-O arabinofuranoside have also been isolated (Liu et al. 2004).

Several flavonoids (quercetin 3-galactoside, quercetin 3-arabinoside, quercetin 3-xyloside, quercetin 3- rhamnoside and two other partially identified 3-pentosides of quercetin and kaempferol in walnut cultivars, collected at different times from walnut grown in Portugal, were also detected (Amaral et al. 2004). Quantification of flavonoid compounds in the walnut grown in Portuguese cultivars performed by HPLC-DAD, revealed that flavonols were always the major compounds, varying between 54.8% and 62.9% of total phenolics, whereas quercetin 3-galactoside was always the major constituent (Pereira et al. 2007). According to the French Pharmacopoeia (10th Ed.) Juglandis folium contains not less than 2% of total flavonoids (Bruneton 1995, Ph. Fr. X).

Phenolic acids/phenolic composition

Previous data revealed the presence of phenolic acids: p-hydroxybenzoic, vanillic, genistic, protocatechuic, p-coumaric, caffeic, ferulic, gallic, chlorogenic (3-caffeoylquinic) acids (Łuczak et al. 1989) and neochlorogenic (5-caffeoylquinic acid) (Blumenthal 2000). Data concerning the quality of walnut leaves samples from six different cultivars grown in Portugal did not confirm the presence of above compounds with the exception of chlorogenic acid (Amaral et al. 2004), which was the major phenolic acid (7.17 – 22.3%), together with 3-O-coumaroylquinic and 4-O-coumaroylquinic acids (Amaral et al. 2004). Moreover a recent publication of the same group described significant differences in the phenolics composition from nine different cultivars over three consecutive years (3- caffeoylquinic, 3-p-coumaroylquinic and 4-p-coumaroylquinic acids, quercetin 3-galactoside, quercetin 3-arabinoside, quercetin 3-xyloside, quercetin 3-rhamnoside, a quercetin 3-pentoside derivative and a kaempferol 3-pentoside derivative) (Amaral et al. 2008). According to the authors suggestion the differences observed can be related to climatic conditions.

Additionally the presence of neochlorogenic and p-coumaric acids in the analysed cultivars were detected; all samples exhibited the same phenolics profile, whereas 3-caffeoylquinic acid was the major constituent (about 19.7%) and p-coumaric acid was the minor compound, representing ca. 1.4% of total phenolics (Pereira et al. 2007).

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).

Volatile oil

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: (E)-β- ocimene (12%), βpinene (11%), limonene (10%), with traces of sabinene, αpinene, myrcene, and linalool, whereas sesquiterpenes by caryophyllene (15%), germacrene D (13%), with minor amounts of (E)-βfarnesene and αfarnesene (Buttery et al. 1986).

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 – β-eudesmol.

Liu et al. (2004) performed an analysis of an essential oil extracted from Juglans regia leaves by hydrodistillation. By use of GC-MS they identified 20 components: terpenoids (84.89%), aromatics (3.9%) and esters (1.34%).

Farag (2008) described detailed results of volatile compounds in the leaves from selected plants of the Juglandaceae family. In Juglans regia, using GC-MS, they identified sesquiterpenes (47%), with germacrene D as the highest concentrated (28%) among them. High levels of monoterpenes were also found in walnut leaves (24%) as well as a high amount of methyl salicylate (17%).

Other compounds

Ascorbic acid (0.85-1%) (Wichtl 2004).

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).

Herbal preparation(s)

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

Topical use

*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 10-50% of well-established use doses when in parallel the same preparations were authorised under well-established use.

**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 pseudo-fructus, 0.35 g Mentahae pip. Folium, 0.35 g Juglandis folium, 0.35 g Graminis rhizoma/ 1 tablespoon [3.5 g]) (Since 1988)

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 pseudo-fructus, 0.105 g Frangulae cortex/ 1 tablespoon [3.5 g]) (Since 1988)

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 anti-inflammatory remedy (Leporatti and Ivancheva 2003).

The leaves of Juglans regia containing repellent and anti-parasitic substances were placed in wheat or in lentils, and in sacks containing other cereals or legumes. The leaves were placed in hen-coops to remove lice. A decoction of leaves was administered to horses as vermifuge and was used to flavour and to preserve cheese in central Italy. In southern Italy, fresh leaves were also applied topically to reduce the swelling associated with varicose veins (Quave et al. 2008). For treatment of burns and to remove warts and pimples, poultices and ointments were applied (Guarrera 1999; 2003; 2005a; 2005b).

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 Indo-Pakistan subcontinent (Akhtar et al. 2000).

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 south-eastern Morocco and in Egypt, Juglans regia leaves and bark are used in oral hygiene, on skin infections and rashes and for hypertension treatment (El-Hilaly et al. 2003; Gazi 1986; Mouhajir et al. 2001; Tahraoui et al. 2007).

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 anti-inflammatory, astringent and depurative. However in the past they were used as anthelmintic, treatment of dyspepsia, diarrhoea, chronic coughs and asthma. The leaves are also used to treat skin disorders: acne (Magin et al. 2006; Reuter et al. 2010), eczema, herpes, scrofulous disease, in excessive perspiration of the hands and feet and slow healing wounds as a wash, compress or poultices). Its astringent activity is assigned to the tannin content (Ożarowski 1976; Ożarowski and Jaroniewski 1987; Blumenthal 2000, Wren 1975,

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.

Assessors’ comment

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

1 week.

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

Deutscher Arzneimittel-Codex (2009)

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)

Daily dose:

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 –6 g of the comminuted herbal substance in 200 ml of boiling water. Apply as an impregnated dressing to the affected areas of the skin 2 – 4 times daily.

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. Non-Clinical Data

3.1. Overview of available pharmacological data regarding the herbal substance(s), herbal preparation(s) and relevant constituents thereof

In vitro experiments

Antioxidant activity

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 (ONOO)]. The walnut leaves extract presented strong antioxidant effects against all studied reactive species with inhibitory concentrations (IC50) in the range of the μg/ml level. Values of IC50 for the ROS O2 and H2O2 were 47.6 ± 4.6, 383 ± 17 μg/ml, respectively. The IC50s for NO and ONOOwere 1.95 ± 0.29 and 1.66 ± 0.10 μg/ml, respectively. Value acquired for ROO.(the oxygen radical absorbance capacity) was 2.17±0.22 μmol

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 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH) radical. The methanol extract was the most effective DPPH radical scavenger with an inhibition of 67.8±0.2%, ethyl acetate and acetone extracts induced 33.9±2.3 and 25.3±1.6% of inhibition, respectively.

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 2-4 independent experiments

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 2-4 independent experiments

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 gram-negative (Escherichia coli, Pseudomonas aeruginosa) gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis) and fungi (Candida albicans, Candida galabrata,

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)

(-): no inhibition zone

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 g-synthase (HpCGS), malonyl-CoA:acyl carrier protein transacylase (HpFabD), and b hydroxyacyl -ACP dehydratase (HpFabZ). Inhibition of HpCGS was non-competitive (IC50 value: 7.0±0.7 μmol/l); inhibition of HpFabZ was uncompetitive (IC50 = 20±1 μmol/l) and inhibition of HpFabZ was competitive (IC50=30±4 μmol/l). The authors concluded that juglone acts as a multi-targeted inhibitor and therefore an encouraging agent against bacterial resistance development.

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 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radicals (EC50=0.151 mg/ml) and the β-carotene linoleate bleaching model (EC50=0.742 mg/ml). These results were much better than those acquired for control standard substances – α- tocopherol and 2-tert-butyl-4-methoxyphenol (BHA). In conclusion the authors suggest the therapeutic use of walnut leaves extract against human gastrointestinal and respiratory infections.

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 MeOH-H2O 1:1 (12 l). The tested extract was eluted with acetone H2O 7:3 (5 l) and freeze dried (7 g). The antibacterial activity of the extract was determined by the disk diffusion method. Zones of inhibition were significantly higher than control standard tea tree oil effects and ranged from 15.8 – 17.6 mm against Propionibacterium acne, 11.3 – 15.7 mm against Staphylococcus aureus and 12.9 – 15.5 mm against Staphylococcus epidermidis. However, the effects were minor when compared to the effects received from doxycycline or clindamycin. Still the authors supported a beneficial role of Juglans regia in acne treatment.

Cruz-Vega et al. (2008) tested in vitro by a microplate dilution assay antimycobacterial the effects of various Mexican plants against Mycobacterium tuberculosis strain H37Rv. Hexane, methanol, ethanol and aqueous extracts of Juglans regia leaves were used. For hexane and methanol extracts, 60 g of leaves were extracted (8.5% w/v), for ethanol – 60 g of the material was macerated with ethanol

(10% w/v) at room temperature, the aqueous extract was obtained by decoction of 30 g of material (6% w/v) and then freeze-dried. Hexane, methanol and ethanol extracts were concentrated under vacuum to yield dry residues. All extracts were stored in the dark at 40C until use. The results are shown in Table 4.

Table 4. Antimycobacterial activity of Juglans regia extracts (after Cruz-Vega et al., 2008)

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 (Ali-Shtayeh and Abu Ghdeib 1999).

Table 5. Mycelial inhibition of tinea capitis dermatophytes (after Ali-Shtayeh and Abu Ghdeib 1999).

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.

Antiviral activity

Min et al. (2002) reported the antiviral activity of quinones, juglone included, on RNase H activity associated with HIV-1 reverse transcriptase. The effect of juglone was moderate and the percentage of inhibition after applying the concentration of 100 μm of juglone was 51.4±1.0 (in triplicate) with an IC50value of 95 μm. However strong inhibitory effects of juglone were seen on the RNA-dependent polymerase (% inhibition=95.5±0.5 with IC50 =8 μm), the DNA-dependent polymerase (% inhibition=99.9±0.0 with IC50 =5 μm) and the DNA polymerase I activity (% inhibition=70.3±1.7 with IC50 =10 μm).

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 non-cytotoxic concentrations. Viruses tested represent models for skin infections (HSV), gastro intestinal infections (polio) and mosquito borne infections (SINV). The results of experiment are shown in the Table 6.

Table 6. Antiviral activity of Juglans regia methanol extract (MIC μg/ml) (after Mouhajir et al. 2001).















Juglans regia












Light and dark activities

Anticancer activity

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). Freeze-dried plant material (1.5 g) was mixed with methanol or petroleum ether (3 x 25 ml). The extracts were filtered and concentrated to dryness under reduced pressure (400C). The methanolic and petroleum ether extraction efficiency in relation to dry matter was 27.7% and 1.1%, respectively. In vitro tests showed also significantly different phenolic contents: 94.39±5.6 and 27.92±1.4 (mg GAE/g extract), EC50 values for the DPPH free radical scavenging capacity: 0.199±0.023 and 2.921±0.740 (mg/ml) ant IC50 values for the antihemolytic activity: 0.06±0.005 (mg/ml) and no activity respectively.

Antiproliferative activity (IC50, mg of methanolic extract/ml) of the Juglans regia leaves towards cultured A-498, 769-P and Caco-2 cells were: 0.226±0.053; 0.352±0.055 and 0.229±0.014, respectively. Values were presented as mean±SEM of three independent experiments, performed in triplicate. The results suggest antihemolytic and antiproliferative effects in human renal cancer cells. .

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 juglone-caused arylation of urease thiol groups is responsible for this effect.

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 skin-whitening agents.

Vasorelaxant Activity

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

Antidiabetic activity

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 alloxan-induced diabetes in rats. There was no difference between Juglans regia extract (200 mg/kg) when compared with antidiabetic activity of glibenclamide 0.6 mg/kg. Both treatments tended to bring the levels of glucose, insulin and glycosylated hemoglobin to near normal. Histological observations showed that the size of Langerhans islets enlarged significantly as compared to diabetic control rats with no treatment.

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.

Anticancer activity

Inhibitory effects of juglone on azoxymethane-induced intestinal carcinogenesis were reported by Sugie et al. (1998). At the start of the experiment, male F344 rats, 5 weeks of age, received orally 200 ppm of juglone in a diet, the control group received only diet. Some groups of rats were receiving once a week for 3 weeks s.c. injections of azoxymetane (AOM, 15 mg/kg bw) to induce tumorigenesis in control rats or together with juglone. After autopsy the number and size of tumors were measured. Juglone treatment decreased the incidence (% animals with tumors) and multiplicity (number of tumors/animals) in small (and entire intestine compared to the group with AOM alone (p<0.05 in each). The maximum diameter of tumors was smaller than that of rats given AOM alone (p<0.05).

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).

Insecticidal activity

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.

Juglone pharmacokinetics

Juglone (5-hydroxy-1,4-naphtoquinone) is present in roots, nut hulls, bark, wood and leaves of the European walnut (Juglans regia). It is a reactive compound due to its redox activity and electrophilicity. Metabolic reduction of juglone involves generation of hydroquinone and also an enzyme-catalysed conjugation of the hydroxyl groups with glucuronide or sulfate. The glucuronides and sulfate conjugates remain in a reduced form and that is why it results in the detoxification of quinones. In the study by Chen et al. (2005) in male F344 rats the disposition of 14C-labelled juglone (0.1 – 10 mg/kg) administered by oral, intravenous and dermal (topical) route was determined (Table 8.).

Table 8. Disposition of juglone-derived radioactivity (% of dose ± SD) in male F344 rats

(n=4) 24 or 72 h following administration. (after Chen et al. 2005)


0.1 mg kg-1,

1 mg kg-1,

1 mg kg-1,

10 mg kg-1,

0.1 mg kg-1,

4 mg kg-1,

4 mg kg-1,


oral, 24h

oral, 24h

oral, 72h

oral, 24h

i.v., 24h






















18.2 ± 3.6

21.3 ± 1.6

23.6 ± 2.8


± 7.2a

32.8 ± 2.5

2.5 ± 0.7

6.3 ± 0.6


63.2 ± 6.2

65.0 ± 3.6

69.2 ± 3.5


± 7.3a

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










C02 and




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

dosing site










89.2 ± 2.1

93.6 ± 3.5

93.6 ± 2.1


± 2.0

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 3H-juglone (0.02 mg/kg, i.v.) were studied by Aithal et al. (2011) in C57/BL mice. After the bolus dose about 35% of juglone accumulated within 15 min in the kidneys, with a half-life of about 2 h.

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

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 7-day treatment of 1 mg/kg juglone of mice, the histological changes were seen in the kidneys, but not in the liver, the spleen and the heart (Aithal et al. 2011).

Ichthyotoxic activity

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).

Prooxidant activity

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 8-hydroxy-2’-deoxyguanozine (8-OHdg), the base adduct in DNA. Moreover juglone formed semiquinone radicals in the presence of ferrous ions.

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 (HL-60), melanoma (MDA-MB435), brain (SF-295) and colon (HCT-8) human cancer cell lines and against peripheral blood mononuclear cells (PMBC) as the control normal cell lines. The cytotoxicity of all compounds was tested using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT). Juglone was dissolved in DMSO 1% and was added to each well and incubated for 72 h.

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 HCT-8 and HL-60 and/or MDAMB435, respectively. (Table 9. Montenegro et al. 2010)

Table 9. Cytotoxic activity of juglone and its derivatives on tumor cell lines, on peripheral blood mononuclear cells (Montenegro et al. 2010)















>5 (28.7)

5 (28.7)



1.33 (7.6)

5 (28.7)








0.02 (0.03)

0.48 (0.8)




0.97 (1.7)







*Data are presented as IC50 values and 95% of confidence interval for leukaemia (HL-60), melanoma (MDAMB-435), nervous system (SF-295), colon (HCT-8) cancer cells and peripheral blood mononuclear cells (PBMC). Dox was used as a positive control. Experiments were performed in triplicate.

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 dose-dependent increase in epidermal ODC activity in the dose range of 880-3250 nmol/mouse. It also induced promotion of papilloma formation (1760 nmol) with the highest response determined with a three times/week topical application. Moreover, juglone lightly converted papillomas to carcinomas (carcinoma/papilloma ratio of 0.35).

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 post-mitotic dephosphorylation and the exit of mitosis. Juglone affected cells had disturbed microtubule dynamics, showed loss of tubulin and formation of tubulin aggregates (Fila et al. 2008).

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 selenium-compromised forms of TrxR (SecTRAPs). TrxR-derived proteins action involves induction of caspase activation that cannot be prevented by Bcl2. SecTRAPs are devoid of thioredoxin reductase activity but can induce rapid cell death in cultured human A549 and HeLa cancer cell lines (Anestål et al. 2008).

In colonies of human cancer cells, juglone induced apoptosis: it was found strongly cytotoxic and induced caspase-3/7 activation in HeLa cells, moreover induced apoptosis in Bcl2 overexpressing HeLa cells and in A549 cells (Cenas et al. 2006). .

In another model of cytotoxicity, juglone inhibited growth and induced apoptosis in human gastric cancer SGC-7901 cells. It was shown that in the range of 5 μm to 20 μm juglone activated caspase 3 in a concentration-dependent manner, Bcl-2 protein expression was down-regulated, level of the ROS generation was increased and up-regulation of Bax protein expression was seen.

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 SGC-7901 cells (24h) (mean±SD, n=3)

(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 S-9 mix was found positive in strain TA2637 and negative in TA98 and TA100 (Tikkanen et al. 1983; Edenharder and Tang 1997).

Tests with S. typhimurium without S-9 mix have given negative results: strains TA98, TA100, TA2637 (Tikkanen et al. 1983; Edenharder and Tang 1997; Juglone NTP http://ntp.niehs.nih.gov/go/19336 ).

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- 9-dependent mutagen 2-AF (Arora et al. 2005). The authors conclude, that the plant extract can be mutagenic as well as antimutagenic depending on the test system used.

No published data could be found on the genotoxicity of European walnut leaves.

3.4. Overall conclusions on non-clinical data

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 anti-inflammatory activity. Results of antimicrobial in vitro evaluations with Juglans regia extracts support the traditional use of European walnut.

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 dose-response studies.

4.2.2. Clinical studies (case studies and clinical trials)

No studies were published on mono-preparations of European walnut.

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

None reported.

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 year-old baker making walnut cakes. Prick testing confirmed flare reactions to walnut (Mendonca et al. 2005).


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.

Drug interactions

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.


None reported.

Effect on ability to drive or operate machinery or impairment of mental ability

None reported

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.

Benefit/Risk Assessment

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.


List of references