Anti-inflammation Activities of Essential Oil and Anti-microbial Activities Of Ethanol Extraction from China’s Rosemary

Jingbo ZHOU,Ruqiang HUANG*,Jihong HUANG,Junwei FENG

1.School of Life Science,South China Normal University,Guangzhou 510631,China;

2.College of Biological Engineering,Henan University of Technology,Zhengzhou 450053,China

Anti-inflammation Activities of Essential Oil and Anti-microbial Activities Of Ethanol Extraction from China’s Rosemary

Jingbo ZHOU1,Ruqiang HUANG1*,Jihong HUANG2,Junwei FENG2

1.School of Life Science,South China Normal University,Guangzhou 510631,China;

2.College of Biological Engineering,Henan University of Technology,Zhengzhou 450053,China

Rosemary(Rosmarius officinalis L.),an endemic plant species in south region of China,is traditionally used as a spice.In this research,the anti-inflammatory activities of essential oil and the antibacterial activities of ethanol extraction were determined,respectively.Results showed that based on the GC-MS analysis there were 35 kinds of active ingredients in the essential oil in totally,mainly including D-limonene(24.158 ml/L),α-Pinene(23.325 ml/L),Camphor(9.855 ml/L), Camphene(7.076 ml/L),Verbenone(6.685 ml/L),Borneol(5.580 ml/L),etc.The LCUV determination indicated that the main components in the ethanol extractionwere rosmarinic acid(3 910 mg/kg)and carnosic acid(2 970 mg/kg).By mice peritoneal macrophage phagocytosis of chicken erythrocytes experiment,the essential oil of rosemary was shown having a significant role in anti-inflammation.And the ethanol extraction had broad-spectrum antibacterial effects,but had no effect on mold by the agar diffusion method of 8 bacteria.As a result,both rosemary essential oil and ethanol extraction had good potential medicinal values.

China’s rosemary;Essential oil;Extraction;Anti-inflammation activity; Anti-microbial activity

R osemary(Rosmarius officinalis L.),a plant of Labiatae perennial evergreen shrub,is also called Ann grass oil[1].This plant grows well in relative warm and dry sandy soil but the growth rate is slower.Rosemary contains many active ingredients which can be divided into several categories,including essential oils,terpenoids,flavonoids,organic acids and others[2].Among them,rosemary’s essential oils can be used as raw materials for the production of cosmetics as well as flavoring agents in meat,poultry or beverage[3].Meanwhile,in the field of medical care,they can effectively alleviate stomach cramps caused by indigestion,flatu lence and other symptoms[4].Rosemary is now recognized as a plant of high oxidative effects.The antioxidative organic acids of rosemary are safe and efficient in removing free radicals[5].At present,a lot of researches of rosemary are focused on the essential oils and other active substances possessing the antioxidative and antibacterial properties[6-8].However,few reports have been focused on the anti-inflammatory effects of the rosemary.In this paper,the anti-inflammatory activities of essential oil bymice peritoneal macrophage phagocytosis of chicken erythrocytes experiment and the antibacterial activities of ethanol extraction from rosemary by the agar diffusion method of 8 bacteria were examined.

Material and Methods

Plant material

Approximately 10 kg of the aerial parts(leaves)of rosemary from Yuzhou,Henan province(China)were collected(in August 2011)during the flowering stage.These plants were identified by manager Han,director of the Biotechnology Company in Henan province.

Preparation of essential oil

The rosemary leaves(100 g)and 1 000 ml distilled water were subjected to steam distillation[9]in a Clevengertype apparatus(RAC EXPORTS,Flask Cap.1 000 ml,India)for 3 h.The essential oil was separated by an oil-water separator(XIANGYI,DL5Y,Hunan).Finally oil was dried over anhydrous sodium sulfate[10].

Preparation of ethanol extraction

The rosemary leaves(100 g) were extracted with ethanol in a constant reflux device(WIGGENS,RX/ RJ,Germany)in order to obtain crude product,and then decolorized,concentrated,washing by hot water,filtered and finally dried[11].

Analysis of essential oil

Analysis of the volatile constituents of essential oil was determined by gas chromatography-mass spectrometry(GC-MS)(Agilent 7890a system,Agilent Technologies)[12].The GC-MS conditions were shown in Table 1.

Determination of ethanol extraction

The ethanol extraction was determined by Liquid chromatography-ultraviolet(LC-UV100S,Shanghai scientific instrument co.,LTD)method.Rosmarinic acid and carnosic acid were used as standard substances,respec tively[13].The testing conditions were shown in Table 2.All determinations were carried out in triplicate and the results were averaged.

Determination of anti-inflammatory activities

The anti-inflammatory activities of essential oil were studied by the immunomodulatory effects-mice peritoneal macrophage phagocytosis ofchicken erythrocytes experiment.In brief,40 Kunming mice(20 males and 20 females,weighing(20±2)g,SPF level,Henan Medical Experimental Animal Center,certification number: 0008328),which were randomly divided into four groups(equality of male and female),were fed for five days,in order to adapt to the environment. Dose groups were subjected to intragastric administration of essential oil (0.27 g/ml)of 0.1,0.2 and 0.3 ml based on every 10 g weight(three times a day,interval 8 h).Both control group and dose groups were in normal diet of drinking water.At the next day after the last gavage,each group were injected into enterocoelia with red blood cell of chicken(RBC,collected from unimmunized cock’s Heart)with a concentration of 5×106/ml(1 ml/each mouse)for 30 minutes,and then mouse peritoneal fluid was eluted by normal saline,collected,smeared, fixed(1:1 acetone-formaldehyde),and dyed(Giemsa-Wright’s stains).The number of macrophages chicken erythrocytes and the total number of red blood cells engulfed by macrophages were counted by oil lens.Data were shown as Phagocytic percentage and Phagocytic index.

Table 1 GC-MS Setting Conditions

Table 2 LC-UV Setting Conditions

Phagocytic percentage=(Macrophage chicken erythrocyts/The total number of macrophages)×100%

Phagocytic index=The total number of red blood cells engulfs by macrophages/The total number of macrophages

Determination of antimicrobial activities

The ethanol extraction against 8 bacteria were studied by the agar diffusion method[14-16].The test organisms used in this study were Enterococcus faecalis 1.2024,Vibrio parahaemolyticus 1.1997,Salmonella enteritidis CICC 21482,Staphylococcus aureus 1.2386,Proteus vulgaris CICC 22929, Aspergillus flavus 3.6434,Bacillus subtilis 1.4255,Escherichia coli 1.8732,all of which were obtained from the collection of the Department of Fermentation Research of Institute of Bio-engineering,Henan university of technology.

The test Aspergillus flavus and Vibrio parahaemolyticus were grown in malt extract broth and nutrient broth respectively at 28℃for 24 h.Other micro-organisms were grown in nutrient broth at 37℃for 20 h.All test micro-organisms in nutrient broth or malt extract broth were enumerated using the serial dilution method.Their nal cell concentrations were 1.5×10-7colonyforming units(cfu)/ml.The agar diffusion method was used to detect antimicrobial activity.A 500 μl aliquot of each micro-organism suspension was placed in a flask containing 50 ml of sterile Mueller-Hinton agar or malt extract agar at 43℃and poured into Petri dishes(21 cm2,Growth area). Then the agars were allowed to solidify at 4℃for 1 h.Four equidistant holes were made in each agar using sterile cork borers(0.4 cm diameter).The extraction was diluted in absolute ethanol(Henan Liyan)and sterile water respectively.Aliquots(60 μl)of the extraction at 95,75 g/L concentrations in absolute ethanol were pipetted into the holes.Dissolved 100 mg extraction into 100 ml sterile water and pipetted 30 μl of them into the holes.Absolute ethanol without the extract was used as controls.Agar plates of Vibrio parahaemolyticus and Aspergillus flavus were incubated at 28℃for 16-26 h in inverted position.Agar plates of the other micro-organisms were incubated at 37℃for 20-28 h.The inhibition zones which formed on the medium were measured(mm)at the end of the incubation period.

Results and Discussion

The results of GC-MS analysis of the rosemary essential oil obtained by hydrodistillation were shown in Fig.1. And the chemical constituents of essential oil were shown in Table 3.Thirty-five components were identified.According to the results,the main constituents of the essential oil from aerial parts of China’s rosemary were D-limonene(24.158 mL/L),α-Pinene (23.325 ml/L),Camphor(9.855 ml/L), Camphene(7.076 ml/L),Verbenone (6.685 ml/L),Borneol(5.580 ml/L),etc (Table 3).This is in accordance with the results by others[17].

According to our results,it was found that the major antioxidative phenolic acids were rosemary acid and carnosic acid and that rosemary acid and carnosic acid were the main constituents in different extraction method. The results of LC-UV analysis of rosmarinic acid and carnosic acid were shown in Fig.2 and Fig.3.

According to our calculations(formulas as below),the yield of the essential oil was 23 ml/kg,the yield of the rosmarinic acid and carnosic acid were 3 910 and 2 970 mg/kg,respectively.

Table 3 Chemical Constituents of essential oil

CA(%)=[(A×MS×V×CAS)/(AS× M×VS)]×100%

A:the average peak area of the sample;MS:standard weight(mg);V: the volume of the sample solution(ml); CAS:CA content of the standard;AS: average peak area of the standard;M: weight of the sample(mg);VS:volume of standard solution(ml).

The mice phagocytic percentage and phagocytic index of different groups were shown in Table 4.Results shown that mice of all the dose groupshad higher phagocytic capacity than those of control group.Phagocytic capacity increased with the increase in the amount of gavage.This study indicated that essential oil from rosemary had a significant role in improving immunity.

The average diameters of inhibition zone of rosemary extraction were shown in Table 5.According to Table 5,there was no obvious inhibition zone in control group(absolute ethanol) which indicated that absolute ethanol had no inhibitory effect and it only acted as solvent in this experiment.95 g/L ethanol extraction and 75 g/L ethanol extraction had inhibitory effect on almost all the tested bacteria,with the most significant inhibitory effect onStaphylococcus aureus.However, the extractions had no inhibitory effect on Aspergillus flavus.Among the tested bacteria,gram-positive bacteria (Bacillus subtilis,Staphylococcus aureus),gram-negative bacteria(Proteus vulgaris,Salmonella enteritidis,Escherichia coli)bacillus(Proteus vulgaris,Salmonella enteritidis)and cocci (Staphylococcus aureus)were included.The results indicated that both the 95 g/L ethanol extraction and 75 g/L ethanol extraction had broad-spectrum antibacterial effect,while having no effect on mold.The sterile water group showed that it only had inhibitory effect on Vibrio parahaemolyticus and Proteus vulgaris.

Conclusions

The present study demonstrated that the main constituents of essential oil from China’s rosemary were D-limonene,α-Pinene,Camphor,Camphene,Verbenone and Borneol.The essential oil displayed anti-inflammatory activities,and the ethanol extraction from China’s rosemary had strong antibacterial activities on several bacteria whereas had no anti-mould activities.Both the essential oil and ethanol extraction of rosemary hadgood potential medicinal values.Further work on the mechanism of antimicrobial and anti-inflammatory is significant to the bioactivity research of China’s rosemary.Such work is ongoing.

Table 4 Phagocytic percentage and phagocytic index

Table 5 Antimicrobial activities of rosemary ethanol extraction

Acknowledements

We thank gratefully to Department of Fermentation Research of Institute of Bio-engineering from Henan University of Technology for providing strains and Key Scientific Research Project of Henan province in China (No.102102310027)for financial support.

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Responsible editor:Xiaoxue WANG

Responsible proofreader:Xiaoyan WU

*Corresponding author.E-mail:qiangdoctor@126.com Received:March 3,2015 Accepted:April 16,2015