Identification of Ages and Determination of Paeoniflorin in Roots of Paeonia Lactiflora Pall. from Four Producing Areas Based On

MICROSCOPY RESEARCH AND TECHNIQUE 75:1191–1196 (2012)

Identification of Ages and Determination of Paeoniflorin in Roots of Paeonia lactiflora Pall. From Four Producing Areas Based on Growth Rings

LIANG-PING ZHA, MING-EN CHENG, AND HUA-SHENG PENG* Department of Pharmacy, Anhui University of Traditional Chinese Medicine and Anhui Key Laboratory of Modernized Chinese Materia Medica, Anhui Hefei 230031, China

KEY WORDS Paeonia lactiflora Pall.; Paeoniae radix Alba; age identification; growth ring; annual ring; root; paeoniflorin ABSTRACT Growth rings were used to determine the root age of medicinal Paeonia lactiflora from four producing areas, and their corresponding paeoniflorin content were measured based on the identification of ages. Different P. lactiflora root samples of different ages were collected from the four major growing areas in China: Bozhou, Anhui Province; Pan’an, Zhejiang Province; Zhong- jiang, Sichuan Province; and Heze, Shandong Province. The relationship between the number of growth rings and age was analyzed using hand sections and paraffin sections. The paeoniflorin content in the roots of different P. lactiflora cultivars from different growing areas was measured using high-performance liquid chromatography (HPLC). The growth rings in the P. lactiflora roots were consistent with the age of the plant from Heze, Zhongjiang, Pan’an, whereas that for the P. lactiflora from Bozhou was one less than the age of the plant. The HPLC results show that the paeoniflorin content was highest in P. lactiflora ‘Baihuachuanshaoyao,’ followed by ‘Baihuahangshaoyao,’ ‘Honghuachuanshaoyao,’ and ‘Honghuahangshaoyao,’ ‘Bozhoushaoyao’ had the lowest levels of paeoniflorin. With increasing age, the paeoniflorin in the roots of the different P. lactiflora cultivars slowly declined or remained the same. In summary, the age of the roots of P. lactiflora from different growing areas can be determined using growth rings. The paeoniflorin content in the roots of P. lactiflora is correlated with cultivar and it was slowly declined with increasing age. Microsc. Res.

Tech. 75:1191–1196, 2012. VC 2012 Wiley Periodicals, Inc.

INTRODUCTION growth ring phenomenon more complex in some peren- The age of many trees can be judged using their an- nial herbs. nual rings, which play an important role in revealing Paeonia lactiflora Pall. is an herbaceous perennial, the past climate change and mechanism (Esper et al., and its roots have been used in medically for at least 2002). In recent years, the growth ring phenomenon 2000 years in China (Peng and Wang, 2007). The Chi- was also observed in roots of some dicotyledonous pe- nese Pharmacopoeia (Pharmacopoeia Commission of rennial herbs, known as the ‘‘herbchronology’’ (Dietz the People’s Republic of China, 2010) defines Paeoniae et al., 1997). Currently, herbchronology has received radix Alba as the roots of P. lactiflora that have been increasing attention in studies on climate change and peeled, boiled, and dried Paeoniae radix Alba is derived disturbance ecology (Dietz and Fattorini, 2002; Dietz from different cultivars of P. lactiflora. Currently there et al., 2004; Schweingruber and Poschlod, 2005; Per- are six P. lactiflora cultivars in the four major growing kins et al., 2006). Growth rings have been reported in areas: Bozhou, Anhui Province; Pan’an, Zhejiang Prov- the roots of perennial forbs in the Duolun Grassland, ince; Zhongjiang, Sichuan Province; and Heze, Shan- Inner Mongolia, China (Liu and Zhang, 2007), but dong Province (Zha et al., 2011a). The breeding and these studies are limited to the field of ecology, and the cultivation methods of P. lactiflora vary among the four growth rings of Chinese medicinal plants have been areas. Cultivation of P. lactiflora usually lasts 3–6 rarely reported. Nearly 300 species of perennial herb years, and it can be harvested after 3–6 years of culti- roots are included in the Chinese Pharmacopoeia. The vation(Zha et al., 2011b).The active ingredients in the accumulation of secondary metabolites in medicinal roots of P. lactiflora is total glucosides of paeony (TGP) plant roots is correlated with growth age (Soldati and Tanaka, 1984).Therefore, determining the age of her- *Correspondence to: Hua-Sheng Peng, Department of Pharmacy, Anhui baceous plant roots to investigate the secondary metab- University of Traditional Chinese Medicine and Anhui Key Laboratory of olite accumulation is important in root medicine. How- Modernized Chinese Materia Medica, Anhui Hefei 230031, China. ever, different from wild plants, some commonly used E-mail: [email protected] Traditional Chinese Medicines are distributed in differ- Received 7 January 2012; accepted in revised form 29 February 2012 Contract grant sponsor: National Natural Science Foundation of China; ent climatic zones and different altitudes after the Contract grant number: 30901973 plants were first introduced, and the cultivation meth- DOI 10.1002/jemt.22048 ods are also different. These conditions make the Published online 17 April 2012 in Wiley Online Library (wileyonlinelibrary.com).

VC 2012 WILEY PERIODICALS, INC. 1192 L.-P. ZHA ET AL.

TABLE 1. Experimental P. lactiflora samples Harvest Age (year)/number Growth area Harvest place Taxon time of plant Bozhou City, Shibali Village, Shijiuli Village, Paeonia lactiflora 2009–10 1/8, 2/4, 3/6, 4/6, 5/4 Anhui Province Huatuo Village ‘Bozhoushaoyao’ Heze City, Shandong Xiaoliu Village, Zhu Village, Paeonia lactiflora 2009–10 1/10, 2/5, 3/4, 4/4 Province Dazhu Village ‘Hezeshaoyao’ Pan’an County, Xinwo Village Paeonia lactiflora 2010–5 1/9, 2/8, 3/8, 4/6, 5/5 Zhejiang Province ‘Baihuahangshaoyao’; Paeonia lactiflora ‘Honghuahangshaoyao’ Zhongjiang County, Jifeng Village Paeonia lactiflora 2010–5 1/9, 2/8, 3/8, 4/7 Sichuan Province ‘Baihuachuanshaoyao’; Paeonia lactiflora ‘Honghuahuachuanshaoyao’

(Wu et al., 2009), which has sedative, analgesic, anti- gent was used to develop color, and a scanner was used inflammatory, hepatoprotective properties, as well as to take images. other pharmacologic activities. Paeoniflorin accounts for over 90% of TGP. A number of scholars have con- Statistics of Growth Ring in Roots of P. lactiflora ducted various studies to measure paeoniflorin content in Paeoniae radix Alba, including determining the One centimeter long samples were collected from all paeoniflorin content in P. lactiflora of different ages roots in 1- to 4-years old P. lactiflora root system from from different growing areas, but their results are four major growing areas. The numbers of growth rings inconsistent (Hong et al., 2003; Hu and Liang, 2009; Li in all roots from the root system were observed under a et al., 2005; Meng and Jiang, 2008; Wang and Chen, microscope to analyze the number of growth rings in 2007; Wu et al., 2006). These inconsistencies may be the root system from the four major growing areas. attributed to the inability to clearly identify the age of P. lactiflora roots. Therefore, determining the root age Experimental Determination of Paeoniflorin of P. lactiflora can be a reasonable assessment of the Content relationship between paeoniflorin content and age of P. P. lactiflora processing at the growth area requires lactiflora roots. peeling and boiling in water. The sequences and time of The objective of this article are as follow: (1) to report peeling and water boiling are different in the various the relationship between growth ring in roots of P. lac- growing areas, and these differences affected the tiflora and growing years, and (2) based on the age results of the measurement of paeoniflorin content in identification, elucidate the correlation of P. lactiflora P. lactiflora (Si and Gu, 2004). To reduce the differen- root age and paeoniflorin accumulation in different ces, all samples were processed by direct drying growing areas. method (i.e., no peeling or water boiling). Dried root samples of P. lactiflora were crushed into powder, and MATERIALS AND METHODS screened using a 60-mesh sieve. The equipment used The P. lactiflora specimens from Bozhou, Anhui included the following: Agilent 1100 HPLC, HS3120 Province; Pan’an, Zhejiang Province; Zhongjiang, digital ultrasonic cleaner, chromatography column, Sichuan Province; and Heze, Shandong Province and quartz sub-boiling high purity water extractor. A (Table 1). mobile phase of acetonitrile:water (18:82) was used at a flow rate of 1.0 mL min21, a detection wavelength of Paraffin Sectioning Method 230 nm, column temperature of 258C, and an injection volume of 10 lL; the theoretical plate number was not Fresh roots of P. lactiflora were harvested. The sam- <2,000 as calculated for paeoniflorin. ples were collected 1 cm from the root head, fixed with Preparation of Standard Solutions. An appropri- FAA solution (70% ethanol:formaldehyde:acetic acid 5 ate amount of paeoniflorin standard was accurately 90:5:5), vacuumed, and dehydrated using different weighed and diluted in methanol to 60 lgmL21. alcohol concentrations. Then, the sections were embed- Preparation of the Sample Solution. Dry P. lacti- ded in paraffin. The prepared sample was baked on a flora root powder was accurately weighed, placed in a Leica HI1220 flattening table, and sectioned with a 50 mL volumetric flask, diluted with 35 mL of 50% Leica RM2265 rotary microtome to slice thicknesses of ethanol, ultrasonicated for 30 min, cooled, supple- 10–15 lm The samples were then baked for more than mented with 50% ethanol to the mark, shaken to mix 24 h, deparaffinized, stained with safranin-fast green thoroughly, and filtered to acquire a standard solution. or phloroglucinol HCl reagent, mounted with neutral Linear Relationship Investigation. The standard gum, observed and photographed under an OLYMPUS solution was accurately injected into a liquid chromato- BH-2 optical microscope and an OLYMPUS LG-PS2 graph at 2, 5, 10, 15, 20, and 20 lL. A standard curve stereo microscope. was made using the peak area as the y-axis and standard volume (lg) as the x-axis. The obtained regression Hand Sectioning Method equation was linear: Y 5 99.104X 1 165.81, r 5 0.9991. Samples were collected from 1 cm from the root head The results show that paeoniflorin standard curve has of fresh P. lactiflora roots, at a thickness of about 1 good linearity at concentrations ranging from 0.4509 to mm. A drop of phloroglucinol–hydrochloric acid rea- 2.2545 lg.

Microscopy Research and Technique AGE IDENTIFICATION OF PAEONIA LACTIFLORA PALL 1193

Fig. 1. Paraffin section image, hand section image, and root map affinsection image of Root of P. lactiflora ‘Hezeshaoyao’ for 1 year old. for the root growth rings of P. lactiflora from Heze of different ages. A: F: Paraffinsection image of Root of P. lactiflora ‘Hezeshaoyao’ for 3 Hand section image of Root of P. lactiflora ‘Hezeshaoyao’ for 2 years years old. G: Paraffinsection image of Root of P. lactiflora ‘Heze- old. B: Paraffinsection image of Root of P. lactiflora ‘Hezeshaoyao’ for shaoyao’ for 4 years old. H: Paraffinsection image of Root of P. lacti- 2 years old. C: Root system of P. lactiflora ‘Hezeshaoyao’. D: Hand sec- flora ‘Hezeshaoyao’ for 5 years old. tion image of Root of P. lactiflora ‘Hezeshaoyao’ for 1 year old. E: Par-

Precision Analysis. Exactly 10 lL of the same Measurement Method. A10lL standard solution paeoniflorin standard solution was injected six times and 10 lL sample solution were accurately taken and each time, which indicated an RSD of 0.45% (n 5 6). injected into liquid chromatograph for measurement. Repeatability Analysis. The same paeoniflorin sample solution was accurately taken, processed as RESULTS described for sample solution preparation method, and Growth Rings in Roots of P. lactiflora injected as above chromatographic conditions. The av- From Heze City erage paeoniflorin content in the peony root was 6.24%, There is only one cultivar, P. lactiflora ‘Hezeshaoyao,’ with RSD 5 0.76% (n 5 6). in Heze, Shangdong Province (Zha et al., 2011a) and it

Microscopy Research and Technique 1194 L.-P. ZHA ET AL.

Fig. 2. The number of growth rings in P. lactiflora roots of differ- growth rings in P. lactiflora roots was 1, the rest may be deduced by ent ages from the four producing areas. (The x-axis represents the analogy;0–10 on y-axis were average number of roots which had dif- number of growth rings, whereas the y-axis represents the number of ferent growth rings in the root system. For instance, x-axis was 1, y- roots which had different growth rings in the root system 0-7 on x- axis was 2, that indicated average number of roots which had 1 axis were numbers of growth rings in P. lactiflora roots. For instance, growth rings was 2). 0 expressed growth rings in P. lactiflora roots was 0, while 1 expressed is reproduced using seeds. The root system has an removed when rhizomes are used for cultivation. Thus, obvious main root, adventitious roots, and lateral roots all roots in the root system develop from adventitious (Fig. 1C). The paraffin sections of the P. lactiflora roots. The number of growth rings in root systems that ‘Hezeshaoyao’ of different ages revealed clear growth have grown for 1 year was zero. As root system grew, a rings in the xylem of the secondary roots, and the num- certain number of adventitious roots developed each ber of growth rings in main root was consistent with an year. In biennial root systems, some roots grow for 2 age of 1–5 years (Figs. 1B, 1E, 1F, 1G, and 1H). The years (the number of growth ring is 1) and 1 year (the hand section stained with phloroglucinol HCl also number of growth rings is 0). Therefore, in 3-year-old revealed clear visible growth rings, and the results are root systems, the roots have 2, 1, and 0 growth rings. consistent with the paraffin sections (Figs. 1A and 1D). In 4-year-old root systems, the roots have 3, 2, 1, and 0 Only one main root was present in the root system of P. growth rings. Thus, the age of P. lactiflora ‘Bozhoush- lactiflora ‘Hezeshaoyao’ and the number of roots with aoyao’ root is equal to its growth ring number plus 1. the maximal number of growth rings in the entire system was usually 1. The lateral roots and adventitious roots develop later, and the number of growth ring was Growth Rings in Roots of P. lactiflora From correspondingly lower. However, for some seeds, after Zhongjiang County, Sichuan Province the main root germinates, the rhizomes will also give rise to more adventitious roots in the same year, and There are two cultivars, P. lactiflora ‘Honghuachuan- the number of growth rings in the adventitious roots shaoyao’ and P. lactiflora ‘Baihuachuanshaoyao’ in was same as that in the main root, leading to more Zhongjiang, Sichuan Province (Zha et al., 2011a). Rhi- roots with same maximal number of growth rings in zome propagation is used to cultivate P. lactiflora ‘Boz- the root system. houshaoyao’ wherein roots are removed when cultivated. However, the number of growth rings in the root systems of P. lactiflora ‘Honghuachuanshaoyao’ and Growth Ring in Roots of P. lactiflora ‘Baihuachuanshaoyao’ are different from P. lactiflora From Bozhou City ‘Bozhoushaoyao’. P. lactiflora ‘Honghuachuanshaoyao’ There is only one cultivar, P. lactiflora ‘Bozhoush- and ‘Baihuachuanshaoyao’ grown for 1 year have one aoyao’ in Bozhou, Anhui Province (Zha et al., 2011a), ring. The number of growth rings increased by one and it is propagated using rhizomes. All roots are each year as the root system developed. That is, the

Microscopy Research and Technique AGE IDENTIFICATION OF PAEONIA LACTIFLORA PALL 1195 growth rings of the P. lactiflora from Zhongjiang are annual. Growth Ring in Roots of P. lactiflora From Pan’an County, Zhejiang Province There are two cultivars, P. lactiflora ‘Honghuahang- shaoyao’ and P. lactiflora ‘Baihuahangshaoyao’ in Pan’an, Zhejiang Province (Zha et al., 2011a). Both cultivars are cultivated using rhizome propagation, and some roots are left on the rhizome when cultivated. The growth ring number and age were consistent in most roots in the root system of P. lactiflora cultivars from Pan’an and the growth rings are annual. How- ever, the number of growth rings in a few roots can exceed their ages. These roots are left during cultivation, and are grown for 1–3 years. Figure 2A was the Fig. 3. Changes in paeoniflorin content in the roots of Paeonia lactiflora Pall. with different growing years from different producing statistical analysis of the growth rings in the root sys- areas. tem of P. lactiflora of different ages in Bozhou, B was in Heze, C was in Zhongjiang, and D was in Pan’an. growing areas, regardless of the method propagation Paeoniflorin Accumulation Dynamics in were consistent with the actual age, i.e., the growth P. lactifora Roots With Different Growing Years rings are annual. Through this experiments, especially From Different Producing Areas the hand sections and the phloroglucinol color develop- Based on the age determination method, the ages of ment method, the age of P. lactiflora from the four the P. lactiflora roots were determined at each growth major growing areas can be quickly determined. area and measured the paeoniflorin content in samples The active ingredient in P. lactiflora roots of is TGP with different growing years from Bozhou, Pan’an, and (Wu et al., 2009), which has sedative, analgesic, anti- Zhongjiang (Fig. 3). The results show that the paeoni- inflammatory, hepatoprotective activities, as well as florin content in the roots of P. lactiflora ‘Bozhoush- other pharmacologic properties. Paeoniflorin accounts aoyao’ did not change significantly with increasing age, for over 90% of TGP. Chinese Pharmacopoeia (Pharma- but it declined significantly in other peony cultivars. copoeia Commission of the People’s Republic of China, The paeoniflorin content in the five different cultivars 2010) used paeoniflorin content as an indicator to mon- were significantly different, with the maximal level in itor the quality of Paeoniae radix Alba. A number of roots of ‘Baihuachuanshaoyao,’ followed by ‘Baihua- scholars have conducted various studies to measure hangshaoyao,’ ‘Honghuachuanshaoyao,’ ‘Honghua- paeoniflorin content in peony, including determining hangshaoyao,’ and ‘Bozhoushaoyao.’ the paeoniflorin content in P. lactiflora of different ages from different growing areas, but their results are DISCUSSION inconsistent (Hong et al., 2003; Hu and Liang, 2009; Li The different ages of roots of P. lactiflora at four major et al., 2005; Meng and Jiang, 2008; Wang and Chen, growing areas were identified through the use of paraf- 2007; Wu et al., 2006). These inconsistencies may be fin sections, hand sections, and microscopic observation attributed to the inability to clearly identify the age of methods. The growth ring phenomenon was observed in P. lactiflora roots. Considering the P. lactiflora root sys- roots of P. lactiflora from all the four major growing tem has roots of different ages, the duration of cultiva- areas for the first time, and growth rings can be used to tion of the root system does not represent the age of identify age of P. lactiflora. Based on the age identifica- each root. The methods for P. lactiflora processing are tion, the paeoniflorin content was measured in P. lacti- different among the various growing areas (Si and Gu, flora of different ages at different growing areas. This 2004). In this experiment, based on the identification of addresses previous deficiencies in measuring paeoni- the root age of P. lactiflora, a uniform processing florin content in P. lactiflora of different ages. method was used to measure the paeoniflorin content P. lactiflora is a perennial herb. It germinates and in P. lactiflora of different ages from the different grow- flowers in spring, and withers in winter (Hong et al., ing areas. The results show that the different cultivars 2001). Most roots of P. lactiflora grow to 20–25 cm had significantly different paeoniflorin content, with below the ground surface and are sensitive to surface the highest level in ‘Baihuachuanshaoyao,’ followed by temperature. Among the four major P. lactiflora grow- ‘Baihuahangshaoyao,’ ‘Honghuachuanshaoyao,’ ‘Hon- ing areas, Bozhou and Heze are located in a warm tem- ghuahangshaoyao,’ with the lowest level in ‘Bozhoush- perate region, and Zhongjiang and Pan’an are located aoyao.’ The paeoniflorin content in all P. lactiflora roots in a subtropical region (Zha et al., 2011a,b). The sea- declined with increasing age, except for ‘Bozhoush- sonal climate significantly changes in all four areas aoyao.’ Thus, the paeoniflorin content in P. lactiflora is and the ground temperature regularly changes with related to cultivar and it was slowly declined with the climate, so the vessels in the P. lactiflora roots also increasing age. Given that the age of P. lactiflora roots change regularly to form growth rings. The experimen- can precisely be identified, the derived paeoniflorin tal results show that the roots from the six cultivars in content in P. lactiflora roots with different ages from the four growing areas all had growth rings. The num- different growing areas can be calculated scientifically ber of growth rings in ‘Bozhoushaoyao’ was 1 less than and accurately, which makes up the inadequacies of the actual age, whereas those from the other three previous studies.

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