Introduction

Dendrobium, one of the largest genera in Orchidaceae, having more than 1,100 species identified, is widely distributed throughout Asia, Europe and Australia (Zhang et al. 2003a). In China, the fresh or dried stems of many Dendrobium species are collectively regarded as a famous herbal medicine. Listed as a “superior grade” medicinal herb in “Shen Nong’s Herbal Classic,” which is one of the earliest herbal pharmacopeia in the world, Dendrobium has been used in Chinese medicine for thousands of years for its traditional properties of supplementing the stomach, promoting the production of body fluids, nourishing Yin, and clearing heat (Deng et al. 2002).

Despite 78 species of Dendrobium found in China, thirty of which are currently used under the same Chinese name Shihu, Chinese Pharmacopoeia (2010 edition) has only two monographs of medicinal Dendrobium plants. One is Dendrobii Caulis (Shihu in Chinese), derived from Dendrobium nobile, D. chrysotoxum, D. fimbriatum and other related Dendrobium species. The other is Dendrobii Officinalis Caulis (Tiepi shihu in Chinese), derived from D. officinale (Fig. 1).

Fig. 1
figure 1

The original plants of D. nobile (a), D. chrysotoxum (b), D. fimbriatum (c) and D. officinale (d)

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Chemical studies on Dendrobium plants have been conducted since the 1930s. While alkaloids, aromatic compounds, sesquiterpenoids and polysaccharides have been identified as the main components (Chen and Guo 2001), the chemical profile varies greatly among species and samples collections (Xu et al. 2010b, 2011; Yang et al. 2006c) which in turn makes these Dendrobium species possess diverse bioactivities: Dendrobium polysaccharides exhibit immunomodulatory, antioxidant and hepatoprotective activity; the alkaloids are anti-cataract and neuroprotective, and the aromatic compounds and sesquiterpenoids exert anti-angiogenesis, anti-tumor and anti-mutagenesis effects (Ng et al. 2012).

Therefore, quality control becomes urgent for ensuring the efficacy and safety of Dendrobium in clinical applications. Chinese Pharmacopoeia (2010 edition) recommended only one chemical marker which seems powerless for so many Dendrobium species involved. Furthermore, there are multiple factors affecting the quality, not only species, but also geographic localities, harvest times and processing methods (Xu et al. 2010b). To date, many studies have attempted to develop accurate, sensitive and selective analytical methods for qualitative and quantitative evaluation of Dendrobium materials.

In this paper, the progress in researches of chemistry, bioactivity and quality control of Dendrobium plants is reviewed with some existing problems addressed and suggestions for further study are also proposed.

Chemistry

In the past 80 years, more than forty Dendrobium species have been investigated for their phytochemistry, with a focus on small molecules, such as alkaloids, aromatic compounds and sesquiterpenoids, while researches on biomacromolecules in Dendrobium species started 30 years ago. The results showed that different Dendrobium sources presented unique chemical profiles.

Alkaloids

Alkaloids are the first category of compounds extracted from Dendrobium with confirmed chemical structures. So far, five types of alkaloids with different structural skeletons have been reported from Dendrobium, namely sesquiterpenoids, indolizidine, pyrrolidines, phthalides and imidazoles (Table 1). As shown in Fig. 2a and Table 1, a sesquiterpenoid, also categorized as the dendrobine-type, has been found in six Dendrobium species. Its basic skeleton consists of one picrotoxane-type sesquiterpenoid combined with a five-membered C2–C9-linked N-heterocycle and a C3–C5-linked lactonic ring (Fig. 2a, 1). Various substituent groups, frequently a methyl group, appear on the N atom. In particular, the C2–C9 N-heterocycle and C3–C5 lactonic ring are open in some sesquiterpenoid alkaloids from Dendrobium species (Fig. 2a, 2–4). Indolizidine alkaloids in Dendrobium are formed by mixed-joint multiple substituted piperidine(s) and pyrrolidine with mutual C and N atoms (Fig. 2b, 1–2). Substituent groups, such as methyl, hydroxyl, acetyl and phenyl, are always present on the piperidine(s). Indolizidine alkaloids have only been found in Dendrobium crepidatum and D. primulinum. The structures of pyrrolidine alkaloids, isolated mainly from D. chrysanthum, are simple. Only one or two pyrrolidines linked by di-substituted acetonyl, with some ordinary substituents, such as cinnamoyl, methyl and acetonyl, compose this kind of alkaloid (Fig. 2c). The other two types of alkaloids, phthalides and imidazoles, are rarely found in the four Dendrobium species investigated (Table 1; Fig. 2d, e).

Table 1 Micromolecular compounds in Dendrobium spp.
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Fig. 2
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Chemical skeletons of alkaloids in Dendrobium. a Sesquiterpenoid type; b indolizidine type; c pyrrolidine type; d phthalide type; e imidazole type

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Main aromatics

A large number of aromatic compounds, represented by bibenzyls, phenanthrenes, fluorenones and coumarins, have been reported from Dendrobium.

Bibenzyls are widespread in different Dendrobium species. For example, moscatilin and gigantol have been isolated from nearly twenty species of Dendrobium (Table 1). The structures of the major bibenzyls in Dendrobium are simple and generally consist of a basic framework, bibenzyl, also known as 1,2-diphenylethane, with substituents, always located at the para- and/or meta-positions on the benzene ring C atoms which are substituted by ethyl (Fig. 3a). The substituents are frequently hydroxyl and/or methoxyl, and sometimes phenyl, phenoxyl, phenacetyl and glucosyl. The total number of these groups is between 3 and 6. The structural diversity of bibenzyls in Dendrobium depends on the type, number and position of these substituents. In particular, the two C atoms in ethyl (C7 and C8) can rarely be substituted. Interestingly, none of the bibenzyls in Dendrobium has been found with mono-substitution. The same situation also occurs with phenanthrenes (except for phenanthraquinones) and fluorenones in Dendrobium. The other bibenzyls found in Dendrobium species possess more intricate structural characteristics (Fig. 3b). In these bibenzyl derivatives, one of the two benzene rings in bibenzyl is always combined with an intricately substituted oxygenic (benzo-) heterocycle. This kind of bibenzyl has been found in abundance in D. officinale.

Fig. 3
figure 3

Chemical skeletons of bibenzyls in Dendrobium. a Simple bibenzyls; b intricate bibenzyls

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Most natural phenanthrenes in Dendrobium species appear to be hydroxyl- and/or methoxyl-substituted 9,10-dihydro or dehydro derivatives (Table 1; Fig. 4a). The numbers of hydroxyl and methoxyl moieties are also between 3 and 6, and can be found usually at C2–7 and sometimes at C1, C8 and C9. Furthermore, C4 and C5 in some phenanthrenes in Dendrobium can be linked with a di-substituted ester or methoxyl group to form an oxygen-bearing hexatomic ring or lactonic ring. Additionally, some novel types of 9,10-dehydro and dihydro-phenanthrene combined with miscellaneous substituted pyran or furan have also been isolated and identified from several Dendrobium species (Fig. 4b). Another type of phenanthrenes found in Dendrobium is the group of phenanthraquinones. 1,4-phenanthraquinone and 9,10-dihydro-1,4-phenanthraquinone are frequent skeletons of phenanthraquinones in Dendrobium (Table 1; Fig. 4c).

Fig. 4
figure 4

Chemical skeletons of phenanthrenes in Dendrobium. a Simple phenanthrenes; b intricate phenanthrenes; c phenanthrenequinones

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Fluorenones and coumarins are widespread in Dendrobium species as well. However, the quantities of these two categories of aromatics in Dendrobium are much lower than bibenzyls and phenanthrenes. Universally, C1–8 of the fluorenones in Dendrobium are selectively substituted by 3 to 5 hydroxyls and methoxyls (Fig. 5a). As to coumarins in Dendrobium species, they generally consist of a coumarin skeleton with C5 and C6, sometimes C2, substituents, mostly hydroxyls and methoxyls (Fig. 5b, c). It is worth mentioning that D. densiflorum and D. thyrsiflorum are the richest resources for coumarins.

Fig. 5
figure 5

Chemical skeletons of fluorenones (a) and coumarins (b, c) in Dendrobium

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Sesquiterpenoids

Sesquiterpenoids are also frequently found in Dendrobium species. Unlike aromatics in Dendrobium, which are widely distributed, sesquiterpenoids have been found in only nine Dendrobium species so far, and the majority is only found in D. nobile and D. moniliforme (Table 1). As shown in Table 1, the picrotoxane type is the most common sesquiterpenoid isolated from Dendrobium (Fig. 6a). Similar to sesquiterpenoid alkaloids in Dendrobium, the picrotoxane skeleton is sometimes combined with C2–C9- and/or C3–C5-linked, rarely C5–C7 or C5–C10-linked, lactonic rings (Fig. 6a). General substituents, mostly hydroxyl and/or hydroxymethyl, and occasionally methyl, methoxyl, carboxyl, carbonyl or glycosyl, are found on the picrotoxane-type skeleton. In particular, C5 and C9 in the picrotoxane skeleton can be linked with a carbonyl (Fig. 6a). The alloaromadendrane type, another kind of sesquiterpenoid (Fig. 6b), is also rich in D. nobile and D. moniliforme. Methyl is always attached to the C4 of the alloaromadendrane skeleton while several kinds of substitutions frequently happen at C10 and C11. Furthermore, other types of sesquiterpenoids, such as cyclocopacamphane, cadinene, emmotin and muurolene, have also been found, mainly in D. nobile (Fig. 6c).

Fig. 6
figure 6

Chemical skeletons of sesquiterpenoids in Dendrobium. a Picrotoxane type; b alloaromadendrane type; c other types

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Other small molecules

In addition to the above mentioned characteristic components in Dendrobium, other common types of micro-molecular compounds have also been isolated from many Dendrobium species (Table 1) and most of them are mono-aromatics, lignans, steroids, flavonoids, or anthraquinones.

Polysaccharides

Polysaccharides, a class of carbohydrate consisting of numerous (usually more than ten) monosaccharides linked by glycosidic bonds in branched or unbranched chains, are usually considered as one of the most important active compounds in medicinal plants. Polysaccharides always present with significant amounts in Dendrobium, even representing up to nearly 50% of the total dry weight in some species, e.g. D officinale (Li and Xu 1990), and have been experimentally proven to exert multiple biological properties, such as immuno-modulation, anti-tumor and anti-oxidant activity (Ng et al. 2012).

To date, several kinds of polysaccharides have been isolated and purified from Dendrobium species (Table 2). However, compared with studies on micro-molecular compounds, reports on the chemistry with regard to isolation, purification, and in-depth structural elucidation of the polysaccharides in Dendrobium are limited and only focus on six frequently used species in China, i.e., D. officinale, D. nobile, D. huoshanense, D. aurantiacum var. denneanum, D. moniliforme and D. aphyllum, as summarized in Table 2. As shown in Table 2, there are discrepancies in reports of the molecular weights of the purified polysaccharides from these Dendrobium species. Glucose, mannose and galactose, which are most frequently found, are the three main monosaccharides comprising the polysaccharides isolated from Dendrobium. In addition, other monosaccharides and uronic acids, such as arabinose, rhamonose, xylose and galaturonic acid, also occasionally appeared in the backbone or branched and terminal residues of purified Dendrobium polysaccharides. Moreover, glucosidic bonds in Dendrobium polysaccharides are complicated, mainly including 1 → 4, 1 → 6 or 1 → 3 linkages. Pyranosyls with α- or β-configuration are prevalent in the polysaccharides found in Dendrobium.

Table 2 Polysaccharides isolated and purified from Dendrobium spp.
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Bioactivity

According to traditional Chinese medical theory, Dendrobium is an herbal tonic for supplementing the stomach, promoting the production of body fluids, nourishing Yin, and clearing heat. At present, accumulating studies provide evidence that Dendrobium demonstrates extraordinarily comprehensive bioactivities, involving the immune, nervous, cardiovascular, endocrine, gastrointestinal and urinary systems (Ng et al. 2012).

Nonetheless, bioactivity studies on Dendrobium still suffer from serious problems. All too often, pharmacological effects of Dendrobium extracts or its pure components have been tested and verified only by in vitro experiments. Crucial factors that might directly influence in vivo efficacy, such as bioavailability and pharmacokinetics, can not be considered by in vitro experiments. Thus, to assess the actual activity of Dendrobium, in vivo experiments must be executed. To provide an example, gigantol, a bibenzyl that normally occurs in Dendrobium, has been reported to possess multiple positive effects that correlate with molecular mechanisms according to in vitro experiments, including anti-cataract, anti-tumoral, anti-mutagenic, anti-inflammatory, and antioxidant effects (Wei et al. 2011; Won et al. 2006; Miyazawa et al. 1997; Simmler et al. 2009). However, few in vivo studies have been carried out to further confirm the bioactivity of gigantol.

Secondly, the concentration-effect paradigm, also called the dose–response relationship, of Dendrobium extracts or components should be a factor of concern in evaluating studies. For example, many reports state that purified polysaccharides from different Dendrobium plants present competitive antioxidant activity (Lin et al. 2003). However, the dosages are often amazingly unreasonable. DFHP, a water-soluble polysaccharide isolated from D. fimbriatum, was tested for its in vitro antioxidant activity (Luo and Fan 2011). In the scavenging activity on ABTS assay, DFHP showed a high scavenging effect on ABTS at 3.0 mg/mL, reaching 90.05%, which was close to that of vitamin C in the same concentration (P < 0.05). However, it should be noticed that the scavenging effect on ABTS of vitamin C at 0.25 mg/mL (about 90%) was already close to the level achieved at 3.0 mg/mL, but obviously much stronger than that of DFHP at 0.25 mg/mL (less than 30%). In other words, the scavenging effect on ABTS of DFHP at 3.0 mg/mL was similar with that of vitamin C at just 0.25 mg/mL. Generally, extraordinarily high concentrations of an extract or a natural product can get a “satisfactory” pharmacological response (Gertsch 2009). Unfortunately, the conclusion “the results indicated that DFHP had strong scavenging power for ABTS radicals and should be explored as novel potential antioxidants” was readily obtained when the similar scavenging effects on ABTS of DFHP and vitamin C were compared at a considerably high concentration (3.0 mg/mL). Additionally, beyond the concentration-effect paradigm, the yield of extracts or refined single components in Dendrobium herbal materials needs to be determined and calculated in order to determine potentially effective dosages for whole herbal material or to evaluate the feasibility of using herbal materials as sustainable resources. This issue has been ignored in most related published papers.

Thirdly, apart from pure components, most in vitro and in vivo pharmacological studies on Dendrobium used crude extracts, such as aqueous extracts, alcohol extracts, crude polysaccharides and total alkaloids (Lin et al. 2003). However, the specific bioactive substances in these crude extracts are obscure, and such information is not helpful for development of novel natural products from Dendrobium medicinal plants because it is difficult to standardize the crude extracts. Thus, crude Dendrobium extracts should be further investigated for particular components that are responsible for bioactivity. Furthermore, the interaction between components in crude Dendrobium extracts should also be studied to reveal the scientific basis that multiple components interact to create holistic therapeutic actions in traditional Chinese medicines.

Quality control

Qualitative analysis

Authentication

Due to the distinct chemical components, bioactivities and clinical effects of different Dendrobium species, authentication of Dendrobium is crucial and the first step for implementing its rational administration as a medicine. Traditional morphological and microscopic approaches along with molecular techniques used for Dendrobium authentication have been reviewed in detail (Zhang et al. 2005d). On the other hand, the diverse chemical components of different Dendrobium herbs make it possible to identify and discriminate Dendrobium species by chemical methods. Recently, multiple chromatographic fingerprints, such as high performance liquid chromatography (HPLC) (Zhang et al. 2003b), capillary electrophoresis (CE) (Zha et al. 2009), gas chromatography (GC) (Wang et al. 2011a) and thin-layer chromatography (TLC) (Wang et al. 2003), have been readily exploited for successful identification and discrimination of five Dendrobium species. In addition, some spectroscopic methods such as 1H nuclear magnetic resonance (1H-NMR) (Zhang et al. 2007b), infra-red spectrum (IR) (Li et al. 2009c), near infra-red spectrum (NIR) (Wang et al. 2009a) and ultraviolet spectrum (UV) (Teng et al. 2009) are also being employed for fingerprint analysis and/or discrimination of Dendrobium species.

In general, though extensive work has been conducted for authentication of Dendrobium species, problems still exist. Morphological and microscopic identification is very limited for Dendrobium herbs or processed products with similar macroscopic and anatomical characteristics. Molecular methods are quite effective, but might be not appropriate for routine use owing to the high cost. In spite of the feasibility of using chromatography for routine sample authentication, current and future studies on the development of fingerprint methods for authentication of Dendrobium species should be more comprehensive and be concerned with not only species, but also other factors that could cause chemical inconsistency, such as sample localities, harvesting time, and processing methods. Spectroscopic methods could be used for ordinary discrimination of Dendrobium species, but they are very limited in Dendrobium authentication due to their poor specificity. Hence, abundant exploratory studies are still needed.

Qualitative analysis of polysaccharides

Quality control of polysaccharides remains a challenge because of their complicated structures and macro-molecular mass. Generally, isolation and purification followed by complete structural characterization is the most reliable method for quality evaluation of polysaccharides in medicinal herbs (Table 2). However, this procedure is complex and time consuming. Therefore, over the past 20 years, rapid and convenient methods for qualitative analysis of polysaccharides in Dendrobium have been developed, in which structural information of the investigated polysaccharides could be partially represented in different ways. Pre-column derivatization HPLC (Zhou and Lv 2011), TLC (Huang and Ruan 1997), GC (Luo et al. 2011) and derivatization polyacrylamide gel electrophoresis (PAGE) (Zha et al. 2012) analysis based on the constituent saccharides profiles produced by total or partial acid hydrolysis have been frequently used for characterization and quality control of crude polysaccharides from different Dendrobium herbs. However, the selectivity of acid hydrolysis for different glycosidic bonds is poor, which limits the structural characterization for various polysaccharides. Consequently, several novel methods were established, in which polysaccharides from Dendrobium were selectively hydrolyzed by specific carbohydrases, especially glycosidases, with more moderate conditions. A new “saccharide mapping” based on enzymatic (carbohydrase) digestion and subsequent chromatographic analysis of enzymatic hydrolysate was successfully employed for discrimination of crude polysaccharides from different Dendrobium species as well as the same species grown in different localities (Xu et al. 2011). Analogously, enzymatic fingerprints derived from carbohydrase hydrolysis followed by PAGE analysis were also created for species and locality identification of Dendrobium (Zha et al. 2012). These methods, based on specific glycosidic bonds, provide a different approach to the concise discrimination of polysaccharides from various origins and are helpful for assessing the pharmaceutical or therapeutic quality of polysaccharides in Dendrobium.

Quantitative analysis

Polysaccharides, alkaloids and aromatics have been proven to be largely responsible for the many biological activities of Dendrobium (Ng et al. 2012). Thus, quantitative analysis for the quality control of Dendrobium has mostly focused on these kinds of compounds. To date, a series of analytical methods have been employed and reported to quantify the contents of active components in various Dendrobium species. However, it should be noted that although sesquiterpenoids are also widely distributed in Dendrobium with proved bioactivities, studies on the quantitative analysis of sesquiterpenoids in Dendrobium has not been carried out yet.

Colorimetry and titration

The contents of total alkaloids and polysaccharides in Dendrobium have been determined by colorimetry and potentiometric titration (Li et al. 2002; Sun et al. 2009; Zhang et al. 2001; Zhu et al. 2010a, b). However, these methods, though simple and rapid, are sometimes unreliable due to the effects of uncontrolled experimental conditions (Xu 2001; Hua et al. 2010).

Chromatography

Currently, HPLC coupled with different detectors, such us UV and MS, has become the preferred analytical technique for separation and quantification of markers from complicated Chinese medicinal material extracts, due to its many advantageous features, including high resolution, favourable reproducibility and powerful maneuverability (Liang et al. 2009). HPLC methods for quantitative analysis of Dendrobium are summarized in Table 3. It can be seen that aromatic compounds, e.g., bibenzyls, phenanthrenes, fluorenones and coumarins, and alkaloids are always selected as chemical markers in HPLC quantitative analysis for the quality control of Dendrobium species. UV detection is mostly employed in these methods. Electrospray ionization (ESI)–mass spectrometry (MS) detection is seldomly used in HPLC analysis for further structural elucidation of targeted compounds in the quality control of Dendrobium. However, differing from aromatics with intense absorption in the ultraviolet region, alkaloids of Dendrobium, especially the sesquiterpenoid alkaloids, are extremely weak in ultraviolet absorption due to the absence of conjugated double bonds in their chemical structures. Thus, HPLC–UV methods for quantification of sesquiterpenoid alkaloids of Dendrobium, which are usually poorly sensitive, are actually inappropriate. Some other universal or sensitive approaches, such as evaporative light scattering detection (ELSD) or mass spectrometry, are suggested to be used to improve sensitivity. Additionally, comprehensive analysis, for example, simultaneous determination of multiple bioactive components by HPLC, is also desirable because the “holistic” actions of medicinal herbs are ascribed to complex chemicals. However, so far limited work has been done for the “holistic” quality control of Dendrobium.

Table 3 HPLC applications on quantitative analysis for quality control of Dendrobium spp.
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Ultra-performance liquid chromatography (UPLC), utilizing sub-2 μm particles as solid phase and operating at much higher system pressure than that of HPLC, could perform analyses with higher resolution, greater sensitivity and greater speed with little solvent consumption. So UPLC has been more and more dominant in the area of pharmaceutical analysis, especially in the analysis of traditional Chinese medicines (Liang et al. 2009). Nevertheless, so far, only one study has reported on quantitative analysis for the quality control of Dendrobium by UPLC (Xu et al. 2010b), in which five components of Dendrobium, of three types, were baseline-separated within 6 min. With apparent superiority compared with HPLC with regard to resolution, sensitivity and analytical time, UPLC coupled with multiple detectors, such as UV and MS, should be widely used in qualitative and quantitative analysis for quality evaluation of Dendrobium in the future.

GC and TLC are also repeatedly used for quantitative purposes in quality control of Dendrobium (Cai et al. 2011; Wang and Zhao 1985). But their application is limited since GC is only available for volatile components while TLC quantification is relatively poor in reproducibility, resolution and sensitivity.

In short, numerous methods of qualitative and quantitative analysis for the quality assessment of Dendrobium have been developed. However, due to the extraordinary differences in morphological, microscopic, molecular and chemical characteristics of different Dendrobium herbs, establishing a universal approach for quality evaluation of multiple Dendrobium herbs remains difficult—a goal, not yet a reality.