Lin Ma?Jun-qing Li?Yuan-dong Hu

Determination of berberine in Phellodendron amurense from different sites of Changbai Mountain

Lin Ma?Jun-qing Li?Yuan-dong Hu

Phellodendron amurense has been used for many years as a medical plant in traditional Chinese medicine and has shown great prospect in recent clinical trials for future applications.Berberine is an essential active compound contained in P.amurense.Our objective in this study was to quantify the contentof berberine in P. amurense from sites at different elevations on Changbai Mountain.We collected samples of P.amurense from five different elevations on Changbai Mountain.Berberine in samples was extracted by ultrahigh pressure extraction (UPE).And the quantity was measured by high performance liquid chromatography(HPLC).First,the optimal HPLC conditions for berberine were identified with satisfactory precision(relative standard deviation,RSD 5.6%),good accuracy(relative error,RE3.6%)and good linear relation(R2=0.9998)in the range of 6.576–328.8 mgL-1.Second,the combination of UPE and HPLC methods in quantitative analysis of berberine showed high repeatability(RSD=3.28%),reproducibility(RSD=4.72%),stability(RSD1.27%)and good recovery(99.54%)for real plant materials.Samples from Heilongjiang Province at the lowest elevation contained the highest amount of berberine. Similarly,the lowest amount of berberine was recorded in samples from Changbai Forest Bureau of Jilin Province collected at the highest elevation in this paper.The proposed UPE–HPLC method is simple,reliable and low-cost for quantitative analysis of berberine.Content of berberine in P. amurense varied significantly by site on Changbai Mountain.

Phellodendron amurenseBerberineChangbai MountainAltitudesUltrahigh pressure extractionHigh performance liquid chromatography

Introduction

Common plant species with wide distributions typically accommodate well to a wide range of environmental conditions(Joshi et al.2001).However,across the range of conditions,the individual genotypes might not always perform well(DeWittetal.1998).Genetically responses to latitudinal variation can result in different phenology (Potvin 1986;Weber and Schmid 1998),growth(Sawada etal.1994;Lietal.1998)and sexualreproduction(Aizen and Woodcock 1992;Winn and Gross 1993)in terrestrial species.In general,ecotypes originating from higher elevations display relatively shortstature,early flowering,and low fecundity.These characteristics might result from lower growth rates or shorter developmentalcycles(Winn and Gross 1993;Li et al.1998).The content of active ingredients in plants can also be influenced by environmental conditions.Berberine content has been reported to vary according to elevation and edaphic conditions(Ghimire et al.2006;Andola et al.2010).

Changbai Mountain is also known as Baekdu Mountain (Baekdu san),a volcanic mountain on the border between North Korea and northeast China.Generally,Changbai Mountain includes the eastern mountains in northeast China at 38460–47300N and 121080–130200E.Eastern Jilin Province,Liaoning Province and Heilongjiang Province are included in the administrative region of Changbai Mountain.The highest elevation of Changbai Mountain is 2,691 m in China at Baiyun peak.It is higher than any other mountain in northeast China.

Phellodendron amurense occupies the Korean pine broad-leaved forest on Changbai Mountain.It is an endemic species in Northeast Asia and a category II protected plant in China.P.amurense is a relic species from the Tertiary and representative of the ancient flora.Changbai Mountain region,as the main site oforigin of P.amurense, is a complete temperate forestecosystem.Itis also one of 17 vital areas for conserving biodiversity.Many primary protected plants in China coexist with P.amurense, including Pinus koraiensis,Larix olgensis Henry,Picea koraiensis Nakai,Abies nephrolepis(Trautv.)Maxim., Fraxinus mandshurica Rupr.,Tilia amurensis Rupr.,Taxus cuspidata,P.sylvestriformis(Takenouchi)T.Wang ex Cheng.The Changbai regions supports rich biodiversity.

Phellodendron amurense is a tree species of Changbai Mountain with high economic value.Ithas been used as a medicinal plant in treating various diseases,such as diarrhea or dysentery,jaundice,morbid leucorrhea,swelling pains in the knees and feet,urinary tractinfections and skin infections(Committee of National Pharmacopoeia 2010). P.amurense has important medicinal values and timber value,and there is large marketdemand forthe trees.Thus, in the Changbai Mountain area,P.amurense has been the primary target for logging and has been destroyed by people.

Berberine is an isoquinoline alkaloid commonly applied as a botanic drug in China and other countries.Itis also a major active compound in P.amurense.In recent years, many studies on the antineoplastic effects of berberine have been conducted in many types of cancer cells.The underlying mechanisms of its antineoplastic process have also been investigated(Inoue etal.2005;Issatetal.2006; Narasimhan and Nair 2005).Berberine inhibits the growth of various cancercelllines in vitro ata relatively low IC50. Meanwhile,many other pharmacological benefits of berberine have been reported,including reducing blood cholesterol(Kong et al.2004),anti-inflammatory effects(Kuo et al.2004),therapies for experimental colitis(Zhou and Mineshita 2000),and treatment of diabetes(Zhou et al. 2007).Many recent studies revealed the potential of berberine in future clinical application such as treatment for breast cancer(Refaat et al.2013),liver fibrosis(Li et al. 2014),idiopathic nephrotic syndrome(Hou et al.2013), and polycystic ovary syndrome(An et al.2014).Due to these important pharmaceutical values,the relationship between berberine content and habitat has been investigated in recent years.Harish et al.(2010)studied the variations of berberine content in another medicinal shrub Berberis pseudumbellata in the western Himalaya,as well as habitat-dependent variations of berberine content in B. asiatica in Kumaon,western Himalaya(ibid.).Because of above benefits,berberine has attracted considerable scientific interest in further development as a medicinal plant. Numerous studies have been conducted on the extraction of berberine from plants.Analytical high-speed counter-current chromatography(HSCCC)was developed for systematically selecting and optimizing the separation process of alkaloids from Coptis chinensis Franch(Yang et al. 1998).A non-ionic surfactant was involved in the microwave-assisted extraction of alkaloids from Rhizoma coptidis(Sun and Liu 2008).Ultra-high-pressure extraction (UPE)has been widely applied in pharmaceutics(He et al. 2009).It shortens the processing time and obtains high extraction yields,and causes no adverse side effects on the activity or structure of the bioactive components(Zheng et al.2013;Zhang et al.2012,2013).Nothing that Liao et al.(2012)develop a procedure for the use of UPE to extract berberine from P.amurense Therefore,in the present study,we applied UPE for the extraction of berberine from P.amurense The berberine in extracts of P. amurense were quantified with high performance liquid chromatography(RP–HPLC).

We collected five samples of P.amurense along an elevational gradient on Changbai Mountain for analysis and comparison.Our results mightpromote the utilization of berberine contained in plants.Our work also provides information useful for conserving P.amurense resources and forestbiodiversity.

Materials and methods

Plant materials and reagents

Phellodendron amurense was cultivated in Jilin Province and Heilongjiang Province at different elevations on ChangbaiMountain.Phloem ofsample trees were collected for testing.The sampling locations are described by elevation,latitude and longitude in Fig.1.Berberine was purchased from China Pharmaceutical and Biological Products Inspection(Lot:110713–201212).HPLC grade solvents(acetonitrile,methanoland ethanol)were obtained from Tianjin Kemiou Chemical Reagent Co.Ltd.,Tianjin, China.

Fig.1 The geographic locations of sampling

Fig.2 Experimental apparatus for ultrahigh pressure extraction,1 plunger,2 ultrahigh pressure vessel,3 pressure-conduction media,4 sealed plastic bag,5 plantmaterials and extraction solvent,6 valve,7 pipe,8 supercharger,9 lubricator groove,10 valve

Extraction apparatus and process

A schematic diagram of the laboratory apparatus configured for this study in UPE mode is shown in Fig.2(Liao et al.2012).The apparatus was mainly composed of the following parts:a lubricator groove,a supercharger,a pressure vessel and some valves.In the extraction,the comminuted plant materials and extraction solvents were firstly loaded and enclosed in a sealed plastic bag.Then the mixture was introduced to the pressure vessel.The pressure was quickly raised by the supercharger to reach the set pressure(more than 100 MPa).The pressure was at this level for a set time period.Finally,the pressure was removed over a period of 1-3 s and the extraction process was completed.

Extraction of berberine from Phellodendron amurense by UPE

Phellodendron amurense samples were crushed and sieved through a 24 mesh screen and 2.0 g of the plant materials was loaded in a plastic bag and the extraction solvent was added in proportion(ethanol concentration of 69.1%, liquid–solid ratio of31.3).The plastic bag was sealed after bubbles were removed.The pressure was raised to 243.30 MPa and held there for 120 s.Then the extracts were obtained after pressure was released.The extracts were filtered using 0.22 l m filter membrane and stored at 4C for subsequent experiments(Liao et al.2012).

Quantitative analysis

OurHPLC system was the HP 1100 series(AgilentTechnologies,Waldbronn,Germany)equipped with ChemStationsoftware(AgilentTechnologies).Itcomprises a binary highpressure pump,an online vacuumdegasser,an auto-sampler,a thermostated column compartment and a photodiode array detectorwith a maximumplotin the range of190–800 nm.All separations were carried outon a Hypersil ODS C18 column (4.6 mm 9 250 mm i.d.,5 l m particle size,Dalian Yi Lite, China).The mobile phase wasa mixture ofacetonitrile(A)and 0.3%triethanolamine aqueous solution(B,pH 3.5).The mixtureratio wasA:B=35:65.The flow rate wasmaintained at 1 mLmin-1and the column temperature was 30C. Injection volume was10 l L.The absorbance forthe detection ofberberine wasatthe 265 nmwavelength.Allofthe standard solutions and sample extracts were filtered through a 0.22 l m syringe filter prior to HPLC analysis.The quantities of target compounds were calculated by comparing their peak areas with those of standard solutions.

Validation of the analytical method

The validation of the analytical method was evaluated by many parameters including linearity,limits of detection (LOD)and quantification(LOQ),precision,recovery,analysisrepeatability and stability.Allstatisticalanalyses were carried out using Excelspreadsheets(Microsoft Co.,USA).

Results

Quantitative analysis of berberine

Alkaloids are the primary effective ingredients in medical plants such as P.amurense.Berberine is a vital alkaloid abundantin P.amurense.Asan importantchemicalmarker, ithas been considered to reflectthe quality of P.amurense. The mobile phase was selected based on the polarity of berberine.After severaltrials with various elution systems, we selected the optimal mobile phase as that consisting of acetonitrile and 0.3%triethanolamine aqueous solution. Underthe conditions,the HPLC reference standards and P. amurense extracts are shown in Fig.3.

Linearity,limitof detection and limitof quantification

To determine the migration time and correcting peak area,6 repeated analysesofstandards were carried outunderoptimal HPLC conditions described above.The calibration curve of berberine was linear in a relatively wide concentration range (6.576–328.8 mgL-1).Between peak area(Y)and the concentration of berberine(X,mgL-1),there was good linear regression(Y=21.637X?11.647)with high correlation (R2=0.9998).LODand(LOQ)were defined asthe signal-tonoise ratios(S/Ns)of3 and 10,respectively.According to the calculation,the LODand LOQvaluesofberberine were 0.084 and 0.256 l g,respectively.

Fig.3 Chromatograms of the berberine standard solution(a)and Phellodendron amurense extraction(b);peak 1,berberine

Precision and accuracy

The precision test was evaluated by measuring intra-day and inter-day variability for berberine.The intra-day variability was detected on the same day for 3 concentrations. The inter-day variability was detected on 3 sequentialdays (days 1,3 and 5)also for 3 concentrations.The relative standard deviation(RSD)of both intra-day and inter-day variability was less than 5.6%,indicating high precision of this method(Table 1).Relative error(RE)was involved as an evaluation of accuracy.Intra-day and inter-day accuracies(RE)of berberine were less than 3.6 and 1.8%, respectively(Table 1).These results demonstrated thatthe accuracy and precision of the proposed HPLC method were adequate for quantifying the berberine content of samples.

Repeatability and reproducibility of the combined UPE &HPLC methods

The samples were extracted with UPE and the extracts were analyzed by HPLC.To study the repeatability of the developed UPE–HPLC method,6 herbalsamples from one source were repeatedly extracted and analyzed under experimental conditions.The berberine content was measured and analyzed(Table 2).RSDof the berberine content in the 6 herbal samples was 3.28%(Table 2),indicating high repeatability.Furthermore,the reproducibility was evaluated by comparing the extraction yields of 6 independent extractions performed on 5 consecutive days.

The relative standard deviations ofallobtained extraction yieldswere less than 4.72%(Table 2).The above results on repeatability and reproducibility indicated thatthe combined UPE–HPLC method was accurate,reliable and reproducible. To evaluate the stability of the samples,the same sample solution was stored at 25C and analyzed every 12 h over 2 days.The results demonstrated thatthe RSDfor berberine was1.08%within 24 h and 1.27%within 2 days(Table 2). These results demonstrated that berberine would remain relatively stable in an aqueous solution for atleast2 days.

Recovery

To test the recovery efficiency of the extraction method,a suitable amountofberberine wasspiked into known samples. The standard added sampleswere extracted and analyzed with the above mentioned procedure.The experiments were repeated 6 times at each concentration.After extracting,thesestandard added samples were analyzed with the previously described HPLC method.The quantity of berbrine in each sample was determined with the corresponding calibration curve.Itcalculated thatthe mean recovery yield forberberine was 99.54%with an average RSDof 0.65%(Table 3).The recovery results revealed that this extraction method was adequate and appropriate forthe analysis.From the results of recovery tests,it also showed that the HPLC method was reliable and accurate enough for quantification ofberberine.

Table 1 Intra-day and inter-day variability for the determination of berberine in Phellodendron amurense

Table 2 Repeatability and reproducibility of the developed UPE–HPLC method and stability of the samples

Table 3 Extraction recoveries of spiked analyte

Determination of berberine in five samples of Phellodendron amurense from different elevations

The berberine content of five P.amurense samples was compared between five different elevations(detailed location in Fig.2).After extracting and analyzing with previously described methods,the quantitative results were obtained:No.5[No.2[No.3[No.1[No.4(Table 4), which followed the same order of elevation from lowestto highest.There was wide variation in the content of berberine in samples from different sites.Samples from Heilongjiang Province contained the highest amount of berberine,while those from Changbai Forest Bureau of Jilin Province contained the lowest amount of berberine. The experimental results demonstrated that the UPE–HPLC combined method was accurate and repeatable.

Discussion

Generally,during evolution,plant secondary metabolism and relevant metabolites are the results of reaction andadaptation to varied environmental stresses.The production of secondary metabolites is closely related to environmental factors(including both biotic and abiotic factors).As one of the secondary metabolites,alkaloids have played crucial roles in plantchemicaldefense against insects and herbivores(Lietal.2007).Thus,there mightbe a correlation between alkaloid production and environments.However,the correlation between biology and environment might not be so straightforward,and this requires further exploration.One of the main sources of environmental variation,particularly at continental scales, might result from climate differences.

Climate variation is reflected in worldwide vegetation zones.Environmentalconditions are determined in partby latitude(Walter 1973).It influences the global patterns of plant distribution,due to the species-specific responses of plants to temperature and precipitation(Woodward and Williams 1987;Woodward et al.1990).As elevation increases,solar irradiance and temperature decrease,while summer photoperiod increases.Because these variables impacton many aspects of plantlife,the widely distributed plantspecies express phenotypic variability across latitudes (Lange etal.1981;Berry and Raison 1981).For example, the humidity is relatively lower in higher elevations, resulting in greater water loss and less rainfall,as well as limited available waterforreplacement.Plants can adaptto conditions atdifferent elevations.

In addition to environmental factors,genetic diversity can also cause differences in berberine content.Yan et al. (2006)reported large genetic differentiation between P. amurense populations,and that these might lead to varied ecological adaptability of different individuals in various regions.Berberine,as an isoquinoline alkaloid,is a major active compound in P.amurense.Thus,the correlation between berberine and elevation was revealed in this study.

Our study demonstrated that the content of berberine varied by elevation in samples from different sites on Changbai Mountain.P.amurense from Heilongjiang Province(altitude=323 m,a.s.l.)contained the highest concentration of berberine,compared with samples from other regions of Changbai Mountain.P.amurense with the lowest concentration of berberine was from Changbai Forest Bureau of Jilin Province(elevation=1,100 m, a.s.l.).Harish et al.(2010a)reported that Berberis pseudumbellata and B.asiatica(2010b)growing atlow elevation contained significantly more berberine than did plants from high elevations.Although from our data there seems a directnegative correlation between elevation and berberine content,it is inappropriate to conclude that all plants growing at lower elevations will definitely contain higher berberine contentbecause other geographic factors such as latitude,longitude,and slope might also have influence. Further research is needed to conclude that P.amurense trees growing at higher elevation typically contain lesser quantities of berberine,

Changbai Mountain supports abundant plant resources, most of which have medicinal and economic values.The area is already partly protected in Changbai National Nature Reserve(1,964.65 km2)and part of the range is a UNESCO Man and the Biosphere Reserve.But the area were too smallto meet conservation demands(Tang etal. 2011).In recentyears,forestresources have been severely damaged.Illegal deforestation and excavation occur frequently.The ecologicalenvironmentis destroyed,resulting in fewer birds spreading seeds,which limits the natural regeneration of P.amurense.The Changbai Mountain population of P.amurense faces dangerous(Yan et al. 2006).In situ protection should be considered for population which content high berberine in order to reserve its good medicinal germplasm resources.

AcknowledgmentsThe authors are grateful to Prof.Ning Zhu of Northeast Forestry University and Chengde Gao of Forestry Management Bureau of Changbai.

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3 March 2014/Accepted:5 June 2014/Published online:10 January 2015

Project funding:This study were supported by the 12th five-year National Science and Technology plan of China(2012BAC01B03) and the 111 Project of China(B13007).

The online version is available athttp://www.springerlink.com

Edited by Zhu Hon

Key Laboratory for Silviculture and Conservation of Ministry of Education,Forest College,Beijing Forestry University, Beijing 100083,People’s Republic of China

e-mail:lijq@bjfu.edu.cn

Y.Hu

Landscape Architecture School,Northeast Forestry University, Harbin 150040,People’s Republic of China