Introduction

Cervi Parvum Cornu is a well-known crude drug derived from horizontal slices of the velvet-covered antlers of male Cervus ssp. deer (Kim et al. 2012c). Various results, including memory improvement (Lee et al. 2010) and anti-infective (Dai et al. 2011), anti-arthritic (Kim et al. 2004), anti-allergic (Kuo et al. 2012), and anti-endometriotic (Kim et al. 2012b) effects, have been reported after use of this product. It has been widely used as an ingredient in functional foods.

To date, two types of Cervi Parvum Cornu, derived from C. nippon (sika deer) and C. elaphus (red deer), have been described in the Korean Herbal Pharmacopoeia (Han et al. 1994). The efficacy and price of antlers vary according to the grade and source of the antler slices (Kim et al. 2012c). The grade and origin of antler slices also affect the price of functional foods containing them. In Korea, antlers are mainly imported from Russia, New Zealand, and China. In Korean markets, Russian antlers are considered to be of particularly good quality and command the highest prices. Merchants may lie about the origin of antlers to charge higher prices. Therefore, consumers should be cautious about buying deer antlers or related products. It is difficult to distinguish morphological differences in origin or subspecies of antlers. For this reason, it is necessary to develop a method that can distinguish Russian antlers from other Cervus antlers.

The quality control of deer products depends primarily on macroscopic identification, microscopic examination, and physical and chemical analyses. However, deer products cannot always be evaluated using these methods because of the different technical processes used to prepare the products (Zha et al. 2011). The use of PCR techniques to identify deer products has been extensively investigated. The PCR approach tends to be more specific, sensitive, and applicable even to heat-processed products (Bottero et al. 2003; Dalmasso et al. 2004). The D-loop region of mitochondrial DNA (mtDNA) has been used to classify deer subspecies (Polziehn et al. 1998; Polziehn and Strobeck 1998, 2002). In our previous studies, we used sequences from the D-loop region to differentiate between deer species and Cervus antlers (Kim et al. 2012a, c).

The goal of the present study was to develop a PCR method to differentiate the most expensive Russian antlers from other antlers in the Korean herbal market. First, we performed subspecies identification of 166 Cervus antler samples collected from China, New Zealand, Canada, Korea, and Russia by amplifying the D-loop region of the mtDNA. All Russian antlers were confirmed to be Cervus elaphus sibiricus. Finally, we developed a PCR-based assay to differentiate between C. e. sibiricus antlers and other Cervus antlers.

Materials and methods

Sample collection

We collected 166 Cervus antler samples from various processing plants in China, New Zealand, Canada, Korea, and Russia (Table 1). Voucher samples for the antlers were deposited with the Korea Institute of Oriental Medicine.

Table 1 Details of the 166 Cervus antler samples used in this study
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DNA extraction, amplification, and sequencing

DNA was extracted from the antler slices using the DNeasy Blood & Tissue Kit (Qiagen, Germany) according to the manufacturer’s protocol. Purity of the DNA was confirmed using an ND-1000 spectrophotometer (NanoDrop Tech., USA). Primers CST2 and CST39 (Table 2) were used to amplify the D-loop region of mtDNA from the extracted DNA (Polziehn et al. 1998). The PCR conditions and cloning method were described in a previous study (Kim et al. 2012c). DNA was sequenced using an ABI 3730 DNA Analyzer (Applied Biosystems, USA). The sequences were aligned using DNASIS MAX version 2.05 (MiraiBio, USA) and BioEdit Sequence Alignment Editor version 7.0.9.0 (Ibis Biosciences, USA).

Table 2 Primer sequences used in this study
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Primer design

After sequence alignment of the D-loop region, we designed a 25-mer SF4 primer (forward primer) and 17-mer SR10 primer (reverse primer) to differentiate between C. e. sibiricus and other Cervus species antlers. Primer locations and sequences are shown in Fig. 1 and Table 2, respectively.

Fig. 1
figure 1

Partial sequences of the mitochondrial DNA (mtDNA) control region used to distinguish C. e. sibiricus from other Cervus subspecies in antler samples. Dots represent identical bases to the first sequence. Primer sets are marked by blue square boxes; the sequences are presented in Table 2

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C. e. sibiricus-specific PCR by agarose gel electrophoresis

Primer sets SF4/SR10 and L14724-tm58/H15149-tm58 (Table 2) were used to amplify gene sequences in the mitochondrial D-loop and cytochrome b regions, respectively. PCR amplifications were performed in a 20-μL volume containing 5 U/μL GoTaq® Hot Start polymerase 0.2 μL (Promega, USA), 4 μL of 5× Green GoTaq® Flexi buffer (Promega, USA), 1 μL of dNTP mixture (Takara Bio Inc., Japan), 1.6 μL of 25 mM MgCl2 solution, 1 μL of 20 ng/μL template DNA, 2.4 μL of 10 pmol primer (SF4/SR10), and 0.7 μL of primer set L14724-tm58/H15149-tm58 (Table 2). The PCR conditions were: initial denaturation at 95 °C for 4 min; 21 cycles of denaturation at 95 °C for 30 s, annealing at 56 °C for 30 s, and extension at 72 °C for 30 s; and a final extension at 72 °C for 6 min. PCR was conducted using a C1000™ Thermal Cycler (Bio-Rad, USA). Amplified products were separated in a 2 % (w/v) agarose gel with 1 μL LoadingSTAR (Dynebio, Korea) in 0.5× TBE buffer (45 mM Tris–borate and 1 mM EDTA; pH 8.0) and were analyzed using a U:Genius (Syngene, UK).

Results

Identification of Cervus antlers using mitochondrial D-loop sequence

To confirm that the 166 antlers belonged to Cervus subspecies, we compared the homology of D-loop sequences of the amplified fragments with known nucleotide sequences in the NCBI GenBank database (http://www.ncbi.nlm.nih.gov/genbank/).

Most samples showed 98–99 % identity with the GenBank sequences, except for the 19 samples from Jilin Province, China, which showed 95–96 % identity with C. nippon (accession number AF016974). These samples had similar nucleotide sequences and were divided into two groups; the two identified sequences were deposited in GenBank as C. nippon NIP1 (KF141944) and NIP2 (KF141945).

The 20 samples collected from Xinjiang and other areas in China showed 99 % identity with C. e. bactrianus (AF296822). Two new sequences of C. e. bactrianus were found and deposited as C. e. bactrianus BAT1 (KF141938) and BAT2 (KF141939). Both sequences showed three base insertions and two base variations compared with AF296822. C. e. bactrianus BAT2 (KF141939) showed one additional sequence variation. One Xinjiang sample was similar to C. elaphus (AF016973) (99 % identity).

Almost all of the samples collected in New Zealand were identified as C. elaphus. Twenty-five samples showed 98–99 % identity with C. elaphus (AF016973 = 23 samples, Y08207 = 2 samples). Four new C. elaphus sequences were found and were deposited in GenBank as C. elaphus ELA1–ELA4 (KF153093–96). All newly deposited sequences showed one base insertion, one base deletion, and three base variations compared with AF016973. One sample had 99 % identity with C. e. nelsoni (AF016965) and was identified as C. e. nelsoni.

The Canadian samples represented three deer subspecies, including 24 Canadian samples that showed 99 % identity with C. e. nelsoni (AF016962 = 15 samples, AF016965 = 9 samples). We found two new sequences of C. e. nelsoni, deposited in GenBank as C. e. nelsoni NEL1 (KF153097) and NEL2 (KF153098). Each new sequence showed one base deletion and variation compared with AF016962. In addition, 13 Canadian samples were identified as C. e. manitobensis (AF016955 = 3 samples; AF016957 = 9 samples; AF005199 = 1 sample). Two new sequences of C. e. manitobensis were found and deposited in GenBank as C. e. manitobensis MAN1 and MAN2 (KF153091 and KF153092). Both sequences showed one base deletion and one base variation compared with AF016957. C. e. manitobensis MAN2 (KF153092) showed two additional sequence variations. The final three samples collected in Canada showed 99 % identity with C. e. canadensis (AY970666). Two of these showed three base variations compared with AY970666, and that sequence was deposited as a new C. e. canadensis sequence (KF141943).

The Korean samples also represented three subspecies of deer, including 10 samples that showed 99 % identity with C. elaphus (AF016973), 11 samples showed 99–100 % identity with C. e. canadensis (AY970666), and 10 samples that showed 99 % identity with C. e. nelsoni (AF016962, AF016965, and AF016966). We found two new sequences of C. e. nelsoni in the Korean samples and deposited these in GenBank as C. e. nelsoni NEL3 (KF153099) and NEL4 (KF153100). These new sequences had one more base deletion than C. e. nelsoni NEL1 (KF153097) and NEL2 (KF153098).

Finally, 29 Russian samples showed 99 % identity with C. e. sibiricus (AF058371). Two new sequences of C. e. sibiricus were deposited in GenBank as C. e. sibiricus SIB1 and SIB2 (KF141940 and KF141941). Both sequences showed one base deletion compared with AF058371. C. e. sibiricus SIB2 showed one additional base deletion and seven base variations. All the results of the identification of 166 Cervus subspecies antler samples are summarized in Table 3. The details of BLAST searches for all 166 Cervus antlers are provided in Supplementary Table 1.

Table 3 Identification of 166 Cervus antler samples by using mitochondrial D-loop sequence
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Differentiation of C. e. sibiricus using electrophoresis-based PCR assay

We observed a difference in the D-loop sequences, which we used to design a primer set (SF4 and SR10). This primer set produced a specific 396-bp fragment that amplified only C. e. sibiricus by agarose gel electrophoresis, allowing it to be distinguished from other Russian antler samples (Lanes 1 and 2 in Fig. 2). A 468-bp fragment derived from the cytochrome b gene was used as the internal control to verify the success of amplification in all samples (Fig. 2). These primers were reconfirmed in all 166 samples, including 29 Russian samples (Supplementary Fig. l). Thus, the DNA sequencing analysis verified that all antler samples collected from Russia belonged to C. e. sibiricus.

Fig. 2
figure 2

PCR products generated with C. e. sibiricus-specific primers. M = 100 bp on a DNA size standard ladder

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Discussion

The consumption of antlers in Korea has been increasing steadily each year, concomitant with economic growth, and imports of antlers have also increased. Cervus antlers are primarily imported to Korea from Russia, New Zealand, and China. In Korean markets, the most expensive deer antlers are imported from Russia. However, Rangifer antlers, derived from reindeer (Rangifer tarandus), and Cervus antlers produced in North America are sold illegally in herbal markets as Russian Cervus antlers (Yoo et al. 2010). Both males and females of the genus Rangifer have antlers, whereas only males of the genus Cervus have antlers (Shah et al. 2008). Rangifer antlers are not considered viable drug material because their ash content does not meet the specified minimum value (Korea Food & Drug Administration 2014; Shim et al. 2011). Since 2001, the import of North American deer antlers infected with chronic wasting disease has been banned in Korea.

In the Korean Herbal Pharmacopoeia and Chinese Pharmacopoeia, the definition of the chemical standards of Cervi Parvum Cornu is not complete. Only the appearance, identification, loss of material during drying, and ash content are specified (Chinese Pharmacopoeia Commission 2010; Korea Food & Drug Administration 2014). It remains difficult to identify subspecies using morphological characteristics and sensory evaluation tests. Morphological distinction is particularly difficult when antlers are sold as cuttings. Several studies have attempted to establish standards to evaluate the quality of Cervi Parvum Cornu, and the amounts of free amino acids, ganglioside, and glycosaminoglycan were proposed as the standard (Hong et al. 1991, 1993; Sunwoo and Sim 2001). Despite such efforts, the criteria to evaluate the quality of antlers remain insufficient, and studies of the pharmacological properties of antlers from different subspecies and regions have not been completed.

Therefore, it is necessary to develop a method that precisely differentiates the region of production and the subspecies of deer antlers. The most effective means of determining the subspecies is analysis of the nucleotide sequences of specific gene regions. However, this procedure is costly and it is challenging to obtain results quickly using this method. If a large number of samples must be analyzed, PCR-based methods are more practical and cost-effective.

In recent years, molecular methods, such as PCR-based assays, have been widely used to differentiate between animal species or subspecies. Oh et al. (2000) conducted assays of Korean beef cattle and Holsteins based on SNP allele differentiation of genes related to coat color. Kim et al. (2012c) and Shim et al. (2011) reported a PCR-based assay for differentiating Rangifer and Cervus antlers. Until recently, little research had been conducted on Russian deer. Baranova et al. (2012) investigated the genetic diversity of R. tarandus inhabiting the European part of Russia. ZvychaĬnaia et al. (2011) conducted polymorphism analysis of mtDNA of the Siberian roe deer (Capreolus pygargus) from 23 regions of Russia and Kazakhstan.

To the best of our knowledge, the present study is the first to focus on identifying the subspecies of deer antlers produced in Russia. We developed a rapid and simple method using specific primers to distinguish C. e. sibiricus from other Cervus subspecies by analyzing antler samples, exploiting differences in the nucleotide sequences of the mtDNA. Agarose gel electrophoresis was used to confirm the C. e. sibiricus-specific primers. Our technique provides more accurate and simple subspecies identification. It may be helpful for quality control and detection of Cervi Parvum Cornu and other products derived from deer.