walnut tree ( juglans regia l. ) , belongs to family juglandaceae ( 1 ) .
walnut contains up to 62% - 68% oil , containing a high amount of monounsaturated and polyunsaturated fatty acids ( 2 ) .
walnut leaves are considered as a source of healthcare compounds and are intensely used in traditional medicine to treat venous insufficiency and haemorrhoidal symptoms ; it is also used for its antidiarrheic , antihelminthic , depurative and astringent properties ( 3 - 5 ) .
this valuable tree has a long history of medicinal use to treat a wide range of health complaints .
dry seeds ( nuts ) are very popular and largely consumed as royal food in iran .
in addition , green walnuts , shells , bark , green husks ( epicarps ) , and leaves are used in the cosmetic and pharmaceutical industries ( 6 ) .
the stem bark is reported to be alterative , anthelmintic , astringent , bactericidal , depurative , digestive , diuretic , laxative , detergent , stimulant , tonic and insecticidal ( 7 ) .
walnut oil is a component of dry skin creams , anti - wrinkle and anti - aging products , because it presents moisturizing properties as well as free radical scavenging capacity ( 8) . in an experimental study , treatment of j. regia extracts in the experimental animal samples resulted in a significant decrease in blood glucose , glycosylated hemoglobin , low - density lipoprotein ( ldl ) , triglyceride and total cholesterol and a significant increase in insulin and high - density lipoprotein ( hdl ) level ( 9 ) .
dm as well as its fatal complications is an important cause of death all over the world ( 10 ) . for a very long time , plants have played an important role in the treatment of many chronic diseases , including dm ( 11 ) .
world health organization ( who ) has recommended the evaluation of effective plants for diseases such as diabetes , for which there are few safe modern drugs .
iranian people with diabetes extensively use the extract of walnut and its hydrosol to control their blood sugar ( bs ) . some of these patients are satisfied with this herbal product and tolerate it well .
however , there are few data about its effectiveness and safety ( 12 , 13 ) .
therefore , more studies are required to determine the effects and side effects of walnut on diabetes .
the only study on the effect of walnut extract on glycemic control in patients with type 2 diabetes showed a significant increase of insulin level and decrease of glycosylated hemoglobin ( 13 ) . according to the literature research ,
  for this reason , authors conducted a pilot study on the efficacy and safety of walnut extract ( wle ) in patients with type 1 diabetes .
walnut hydrosol was obtained from a local market in meymand , fars province , iran .
the essential oil of the sample was extracted using a liquid extractor in two stages . at first , 500 ml of the sample was mixed with 500 ml of petroleum - ether as a solvent .
the solvent was heated to 45c for 150 minutes and the essential oil was transformed from an aqueous phase to a petroleum - ether phase .
the remains of the organic phase from the first stage were removed and additional amount ( 500 ml ) of fresh petroleum - ether was added to the system .
this stage was carried out to increase the yield of the essential oil in the organic phase . the extracted essential oil
500 ml of petroleum - ether containing the essential oil was heated to 40c and vacuumed by rotation of a pump motor at 60 rpm , evaporating the petroleum - ether and leaving the extract .
this process produced approximately 50 ml of the sample dissolved in a small amount of organic phase .
the concentrated essential oil was dehydrated and subjected to gas chromatography mass spectrometry ( gc / ms ) for analysis of the constituents ( agilent technologies 7890 gas chromatograph coupled with an agilent technologies model 5975c mass detector , palo alto , ca ) ( 14 ) .
the apparatus was equipped with a hp-5ms capillary column [ phenyl - methylsiloxane , length 30 m  inner diameter 0.25 mm , agilent technologies ( 60 - 325/350c ) ] .
the oven temperature increased from 60c ( 0 minute ) to 220c in increments of 5c / minute and held at that temperature for 10 minutes .
helium was selected as the carrier gas and the flow rate was adjusted at a rate of 1 ml / minute . the mass spectrometer operated in ei mode at 70 ev .
the interface temperature was 280c and the mass range was 30 - 600 m / z .
retention indices were determined by using retention time of n - alkanes injected after the essential oil under the same chromatographic conditions .
the components of the oil were identified by calculation of kovat s indices ( ki ) and comparison of their mass spectra with those of wiley library or the published ones ( 15 , 16 ) . as a pilot study ,
inclusion criteria were type 1diabetes , age over 12 years old , completed puberty , having diabetes for more than two years and use of insulin analogs including lantus solostar pen and novorapid flexpen to control diabetes . subjects who used medicines other than insulin , those afflicted with other associated diseases and those who used insulin types other than lantus solostar pen and novorapid flexpen were excluded from the study .
they were advised to measure their bs level at least four times daily by glucometer and record their bs level and insulin doses for at least two weeks .
then , the patients were advised to drink 250 ml wle after meals twice a day .
they continued to measure and record their bs level and insulin doses . during the study ,
the subjects were called frequently to evaluate compliance and any complications and also to adjust the dosage of insulin injection . after four weeks , the use of wle was stopped , subjects were examined by a physician , and the data including bs levels and injected insulin doses were collected .
the subjects continued measuring and recording their bs levels and insulin doses for two more weeks . at that time
insulin doses and bs levels were compared for each patient before and after consuming wle .
walnut hydrosol was obtained from a local market in meymand , fars province , iran .
the essential oil of the sample was extracted using a liquid extractor in two stages . at first , 500 ml of the sample was mixed with 500 ml of petroleum - ether as a solvent .
the solvent was heated to 45c for 150 minutes and the essential oil was transformed from an aqueous phase to a petroleum - ether phase .
the remains of the organic phase from the first stage were removed and additional amount ( 500 ml ) of fresh petroleum - ether was added to the system .
this stage was carried out to increase the yield of the essential oil in the organic phase .
the extracted essential oil was concentrated by rotary evaporator equipped with a vacuum pump . in this process ,
500 ml of petroleum - ether containing the essential oil was heated to 40c and vacuumed by rotation of a pump motor at 60 rpm , evaporating the petroleum - ether and leaving the extract .
this process produced approximately 50 ml of the sample dissolved in a small amount of organic phase .
the concentrated essential oil was dehydrated and subjected to gas chromatography mass spectrometry ( gc / ms ) for analysis of the constituents ( agilent technologies 7890 gas chromatograph coupled with an agilent technologies model 5975c mass detector , palo alto , ca ) ( 14 ) .
the apparatus was equipped with a hp-5ms capillary column [ phenyl - methylsiloxane , length 30 m  inner diameter 0.25 mm , agilent technologies ( 60 - 325/350c ) ] .
the oven temperature increased from 60c ( 0 minute ) to 220c in increments of 5c / minute and held at that temperature for 10 minutes .
helium was selected as the carrier gas and the flow rate was adjusted at a rate of 1 ml / minute . the mass spectrometer operated in ei mode at 70 ev .
the interface temperature was 280c and the mass range was 30 - 600 m / z .
retention indices were determined by using retention time of n - alkanes injected after the essential oil under the same chromatographic conditions .
the components of the oil were identified by calculation of kovat s indices ( ki ) and comparison of their mass spectra with those of wiley library or the published ones ( 15 , 16 ) .
as a pilot study , eight patients with type 1 dm were enrolled in the study .
inclusion criteria were type 1diabetes , age over 12 years old , completed puberty , having diabetes for more than two years and use of insulin analogs including lantus solostar pen and novorapid flexpen to control diabetes . subjects who used medicines other than insulin , those afflicted with other associated diseases and those who used insulin types other than lantus solostar pen and novorapid flexpen were excluded from the study .
they were advised to measure their bs level at least four times daily by glucometer and record their bs level and insulin doses for at least two weeks .
then , the patients were advised to drink 250 ml wle after meals twice a day .
they continued to measure and record their bs level and insulin doses . during the study ,
the subjects were called frequently to evaluate compliance and any complications and also to adjust the dosage of insulin injection . after four weeks , the use of wle was stopped , subjects were examined by a physician , and the data including bs levels and injected insulin doses were collected .
the subjects continued measuring and recording their bs levels and insulin doses for two more weeks . at that time
insulin doses and bs levels were compared for each patient before and after consuming wle .
the constituents of the essential oil of juglans regia l. are presented in table 1 .
several compounds were identified in the oil and the rate of monoterpenoid and sesquiterpenoid compounds were 53.45% and 5.95% , respectively ; also , the rate of phenolic compound was 51.34% .
the main constituents of the oil were carvacrol ( 33.21% ) , thymol ( 16% ) , and homoveratrole ( 15.83% ) . abbreviations : ki , kovat s indices ;
two of them were male and six were females with the age range of 13 - 25 years , and the mean age of 19.7 years .
table 2 shows the effect of wle on average daily bs level and insulin dose of each subject .
two other subjects showed a decrease only in average daily bs level , but because their bs level was in a favorite range , they did not decrease the insulin doses .
in one other subject , the decrease in the average daily bs level was only 5 mg / dl , which was insignificant .
however , a few days after wle consumption , two subjects called one of the authors and complained about development of generalized pruritic erythematous rashes .
the rashes were mild and non - progressive in one of them ; therefore , she continued wle consumption , but the other subject stopped using wle after two weeks due to progressive and troublesome skin rashes .
she did not have any illness before the attack of hypoglycemia , and overall , there was no other risk factor for hypoglycemia for her .
the level of consciousness was improved in the subject after a few hours and she was discharged from hospital without any side effects .
two weeks after stopping wle use , insulin doses and average bs level of all subjects increased again to nearly the level before using wle .
it includes three species : j. nigra , j. cinerea and j. regia although only j. regia grows in iran ( 17 ) .
the key chemical composition of walnut is juglone ( 5-hydroxy-1 , 4-naphthoquinone ) , the toxic compound which is found only in green and fresh walnuts , but such property disappears in dried leaves ( 18 ) .
several other phenolic compounds with antioxidant properties are identified in j. regia leaves ( 19 ) .
walnut ( juglans regia l. ) is a plant with a significant economic value and medicinal importance for human health .
it is consumed in large quantities by people ; therefore , it has a very important place in the public nutritive habits ( 20 ) .
leaves of j. regia are widely used in folk medicine to treat venous insufficiency and haemorrhoidal symptoms , for their antidiarrheic , antihelmintic , depurative , and astringent properties ; mix of leaves and stored - grains are also used as fungicide and insecticide ( 21 , 22 ) .
other properties such as antibacterial , human cancer cell antiproliferative , antioxidant , keratolytic , antifungal , hypoglycemic , hypotensive , anti - scrofulous and sedative are also reported for this plant ( 19 , 23 - 25 ) .
the beneficial effect of walnut consumption against many diseases is reported , including protection from diabetes ( 26 ) or cardiovascular diseases ( 27 ) .
researches have also shown that eating walnuts can improve the blood lipid profile ( 28 ) .
other investigations showed that j. regia extract contains ellagitannins , which is an anti - cancer agent with anti - inflammatory effects ( 17 ) .
examples are phytochemical analysis of the leaf volatile oil of walnut tree ( 29 ) , studying about protein fractionations , amino acid composition , molecular weight distribution and gel electrophoresis of juglans regia l. ( 30 ) , surveys on antioxidant and antibacterial activities of the leaf essential oil of juglans regia l. and its constituents ( 31 ) , determination of mineral contents of juglans regia l. flowers and its anti - hemolytic activity .
also , another study reported that j. regia improves glycemic level in the patients with type 2 diabetes , without any adverse effects on the kidney and hepatic function ( 14 ) . in another study
( 29 ) , the volatile oils of all 28 j. regia populations were analyzed by gas chromatography flame ionization detectors ( gc / fid ) and gas chromatography mass spectrometry ( gc / ms ) .
major components of the essential oils in this research were ( e)-caryophyllene ( 1.4% - 47.9% ) , -pinene ( 4.5% - 39.5% ) , germacrene - d ( 5.0% - 23.3% ) , -pinene ( 1.5% - 18.1% ) , -humulene ( 1.1% - 11.8% ) , -zingiberene ( 0.1% - 11.3% ) , -copaene ( 0.0% - 10.1% ) , limonene ( 0.8% - 8.6% ) , caryophyllene oxide ( 0.1% - 8.6% ) , ar - curcumene ( 0.0 - 7.2% ) , -cadinene ( 0.3% - 6.7% ) , ( e)--farnesene ( 0.0% - 5.9% ) , 1 , 8-cineole ( < 0.0 - 5.4% ) , -curcumene ( 0.0 - 4.2% ) , methyl salicylate ( 0.1% - 4.0% ) , ( e)-myrtanol acetate ( 0.0 - 3.8% ) , ( e)-muurola-3 , 5-diene ( 0.0 - 3.8% ) , ( e)--ocimene ( 0.6% - 3.9% ) , -longipinene ( 0.0 - 3.0% ) , myrcene ( 0.2% - 2.6% ) , -muurolene ( 0.1% - 2.5% ) , spathulenol ( 0.0 - 2.2% ) and -cadinol ( 0.1% - 2.0% ) ( 29 ) .
a single report on volatile compounds ( head space analysis ) of j. regia leaves from egypt showed the presence of germacrene - d ( 28.6% ) , and methyl salicylate ( 16.8% ) as the main constituents ( 32 ) .
-pinene ( 30.5% ) , -caryophyllene ( 15.5% ) , -pinene ( 15.1% ) , germacrene - d ( 14.4% ) and limonene ( 3.6% ) were identified as the principal components of the essential oil of walnut leaves from kashmir ( 31 ) . in another research ,
the essential oil was obtained by headspace method and the volatile compounds were pentanal ( 0.07 - 0.12% ) , hexanal ( 0.26 - 0.80% ) , nonanal ( 0.34 - 0.89% ) and 2-decenal ( 0.25 - 0.68% ) and hexanol ( 0.21 - 1.58% ) ( 33 ) .
this research indicated that the percentage of aldehyde compound is higher than that of alcoholic compound .
these differences can be due to ecological factors or species variations . according to the current study searches in database , this is the first pilot study to determine the effect of wh on glycemic control in subjects with type 1 diabetes .
previously , only one study was conducted on the effect of walnut extract on patients with type 2 diabetes and several similar studies on animals with diabetes .
wle caused improvement of glycemic control both in rats with diabetes and patients with type 2 diabetes ( 14 , 34 ) .
in the present study , glycemic level controlled in most of the subjects with type 1 diabetes .
they concluded that walnut may cause increased release of insulin from beta cells , increased insulin sensitivity , or may interfere with absorption of dietary carbohydrate ( 34 , 35 ) .
the pathophysiology of t1 dm is completely different from that of type 2 diabetes . in t1
dm , most of beta cells are destroyed ; therefore , many agents that can help patients with type 2 diabetes are not efficacious in t1 dm .
the key compounds responsible for anti - hyperglycemic effect of wle may be phenolic compounds .
phenolic acids and flavonoids are two major groups of phenolic compounds existing in walnut leaves . as it was mentioned , seven components were found in wh . according to the current study investigation on these components ,
therefore , carvacrol or thymol or both of them may have an anti - hyperglycemia role but further studies are needed to investigate the cases . in an animal study , carvacrol in combination with rosiglitazone caused improvement in blood glucose and glycosylated hemoglobin high fat diet induced type 2 diabetes in mice ( 36 ) .
another animal study revealed the anti - hyperglycemic effect of methanol extract of otostegia persica boiss ( labiatae ) on rats with type 1 diabetes .
more extensive studies are needed to determine the exact mechanism of anti - hyperglycemic effect of wle .
walnut extract was not associated with any complications in animals and patients with type 2 diabetes ( 14 , 34 ) , but its side effects were important among the current study subjects .
two of the subjects showed drug reactions as skin rashes , and one of them developed severe hypoglycemia .
drug reaction at least partially may be due to technical problems in providing the extract leading to an impure extract .
therefore , in leading studies , this process should be performed with more caution to prevent adding impurities .
no other etiology was found for hypoglycemia in this subject and , therefore , hypoglycemia was probably the result of wh consumption .
this complication can be an important limiting factor for extended use of the extract in human .
wh may control glycemic level in human , but it can be associated with minor and major side effects .
seven compounds were identified in walnut oil . according to the current study search , among these components two of them ( thymol and carvacrol ) may have an anti - hyperglycemia effect mentioned in other studies .
therefore , wh should not be advised extensively to people with diabetes until the knowledge is increased about its mechanism of action and potential side effects .
the most important limitation of this study was the low number of participants . according to the severe side effects in one of the subjects , it is not suggested to perform this study on larger population .
however , new in vitro studies are recommended to determine the mechanism of wh effect on blood sugar in subjects with type 1 diabetes mellitus .
wh may control glycemic level in human , but it can be associated with minor and major side effects .
seven compounds were identified in walnut oil . according to the current study search , among these components two of them ( thymol and carvacrol ) may have an anti - hyperglycemia effect mentioned in other studies .
therefore , wh should not be advised extensively to people with diabetes until the knowledge is increased about its mechanism of action and potential side effects .
the most important limitation of this study was the low number of participants . according to the severe side effects in one of the subjects , it is not suggested to perform this study on larger population .
however , new in vitro studies are recommended to determine the mechanism of wh effect on blood sugar in subjects with type 1 diabetes mellitus .
 backgroundwalnut hydrosol ( wh ) is used extensively by iranian people with diabetes in order to control blood sugar ( bs ) . 
 there are few data regarding the effect of walnut on controlling diabetes.objectivesa pilot study to determine the efficacy and safety of wh in patients with type 1 diabetes.materials and methodseight patients with diabetes mellitus ( dm ) type 1 were enrolled in the study . 
 they did not use any medicine except insulin . 
 they were advised to drink 250 ml wh after meals twice a day for four weeks . their bs level was measured and their insulin dose was changed according to their bs . after four weeks 
 , they discontinued wh use and their bs level was checked for two weeks . 
 descriptive statistics was used to analyze the data . also , the essential oil of the sample was extracted using a liquid extractor and then analysis of the constituents was performed.resultsthe average daily bs level and insulin dose decreased in seven subjects . 
 two subjects developed generalized pruritic erythematous skin rash . 
 one patient presented hypoglycemic coma . 
 she had no other coma risk factor . 
 seven compounds were identified in the walnut essential oil and the rate of monoterpenoid and sesquiterpenes hydrocarbons were 53.45% and 5.95% , respectively . 
 the main constituents of the oil were carvacrol ( 33.21% ) , thymol ( 16% ) and homoveratrole ( 15.83%).conclusionswh may control the glycemic level in people with diabetes , but it may be associated with minor and major side effects . 
 further in vitro studies , using these seven compounds , are recommended to determine the efficacy and complications of wh in people with diabetes . 
1. Background
2. Objectives
3. Materials and Methods
3.1. Plant Material and Extraction of Essential Oils
3.2. Essential Oil Concentration
3.3. Gas Chromatography-Mass Spectrometry
3.4. Patients
4. Results
5. Discussion
5.1. Conclusions