Age and harvest season affect the phenylpropenoid content in cultivated European Rhodiola rosea L.

Industrial Crops and Products 83 (2016) 787–802

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Age and harvest season affect the phenylpropenoid content in cultivated European Rhodiola rosea L.夽 Wieland Peschel a,∗ , Alfred Kump b , Attila Horváth c , Dezso˝ Csupor c a b c

European Medicines Agency, 30 Churchill Place, Canary Wharf, London E14 5EU, United Kingdom Botanische Arbeitsgemeinschaft am Oberösterreichischen Landesmuseum, Biocenter Linz, J.-W.- Klein Str. 73, 4040 Linz, Austria University of Szeged, Faculty of Pharmacy, Department of Pharmacognosy, Eötvös u. 6, 6720 Szeged, Hungary

a r t i c l e

i n f o

Article history: Received 10 May 2015 Received in revised form 30 September 2015 Accepted 16 October 2015 Available online 29 December 2015 Keywords: Rhodiola rosea L. Authentication Cultivation Standardisation Rosavin Cinnamyl alcohol Phenylpropenoids

a b s t r a c t Characteristic phenylpropenoids are a quality marker to distinguish rhizome and root of authentic Rhodiola rosea L. from other Rhodiola species. A consistent content in line with pharmacopoeial requirements is one objective of increasing cultivation to satisfy the worldwide demand. We set out to compare the influence of harvest season and age on total rosavins (ROStot ) and their aglycon cinnamyl alcohol (CA) determined by HPLC/DAD. Plants from 9 different European origins were grown homogenously in South England and harvested in March, August and November of cultivation years 3–5. For experiment optimisation and validation we initially studied other factors that influence the chemical profile: sample origin (plants, herbal drugs and final products of different origin), plant part (rhizome, root, herb), drying (temperature and duration), extraction (solvent strength). We also investigated differences between plant individuals of the same provenance such as male and female plants. Pre-tests showed the importance of confirmed plant identity as non-authentic samples are indicated by total and relative amounts of phenylpropenoids vis-a-vis phenylethanoids. Rhizomes contained 2–3 times higher ROStot values than roots. There was no substantial influence of drying temperature (45 ◦ C versus 65 ◦ C), but drying at room temperature longer than 10 days influenced negatively phenylpropenoid values. ROStot are best extracted with 70–90% ethanol; CA with 50–70% ethanol. No significant influence of plant sex on the phenylpropenoid content was detected. Extracts (70% ethanol) from R. rosea rhizomes contained 0.5–4.1 mg/mL total rosavins corresponding to 0.31–2.6% in the dry drug. Across all provenances the ROStot and to less extent the CA content in rhizomes was significantly higher when harvested in March than in August or November alongside a decrease from year 3 to year 5 under our cultivation conditions. The CA content was 5–30% of ROStot with some influence of the plant origin and may be considered for drug identification and standardisation. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Rhodiola rosea L. (Crassulaceae) is increasingly cultivated worldwide as a valuable non-food crop to satisfy the demand for rhizome and root (Platikanov and Evstatieva 2008; Ampong-Nyarko et al.,

Abbreviations: CA, cinnamyl alcohol (trans-cinnamic alcohol); DW, dry weight; FW, fresh weight; PPtot , total phenylpropenoids = total rosavins + their aglycon CA; ROStot , total rosavins: rosarin + rosavin + rosin (calculated as rosavin); SALtot , total salidroside = salidroside (calc. as salidroside) + its aglycon tyrosol (calc. as tyrosol). 夽 The views expressed in this article are the personal views of the authors and may not be understood or quoted as being made on behalf of or reflecting the position of the European Medicines Agency or one of its committees or working parties. ∗ Corresponding author at: 30 Churchill Place, Canary Wharf, London E14 5EU, United Kingdom. E-mail address: [email protected] (W. Peschel). http://dx.doi.org/10.1016/j.indcrop.2015.10.037 0926-6690/© 2015 Elsevier B.V. All rights reserved.

2011). The drug is used in products that are sold as adaptogens, anti-stress remedies and with other medicinal claims in different product categories. Popularity goes alongside quality issues mainly due to resource limitations and partially insufficient quality control leading to admixtures or substitutions with other Rhodiola species (Galambosi 2006; Ma et al., 2011). While the role of characteristic constituents for the therapeutic effects is still discussed, phenylpropenoid glycosides (rosavin, rosin and rosarin) are decisive quality markers to distinguish authentic samples of R. rosea from other common Rhodiola species mainly of Asian origin (Ma et al., 2011; Peschel et al., 2013). With increasing cultivation it is the aim to produce material with a high and consistent phenylpropenoid content which is beside salidroside the current marker for standardisation originally based on collected wild plants. For instance the USP monograph requires 0.3% dry weight (DW) of the phenylpropenoid glycosides rosarin, rosavin, and rosin calculated

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Table 1 HPLC validation data for rosavin, cinnamyl alcohol (CA), salidroside and tyrosol.

rosavin CA salidroside tyrosol

R2

Regression equation

Range (␮g/mL)

LOD (␮g/mL)

LOQ (␮g/mL)

r.s.d. (%, n = 5)

0.9965 0.9947 0.9915 0.9994

y = 4E-05x + 0.7058 y = 2E-05x − 30.216 y = 0.0004x + 34.79 y = 0.0002x − 0.9092

2.5–1000 5–5000 10–2500 10–5000

2.6 5.3 27.1 7.2

6.1 13.2 42.7 15.6

3.1 3.7 4.8 5.2

as rosavin (beside 0.08% salidroside); an Australian extract standard not less than 1.8% phenylpropenoids and 1.2% rosavin (beside 0.6% salidroside) (USP 2015; TGA 2014). We previously reported the content of total rosavins and salidroside in liquid extracts from 3-year old cultivated R. rosea of European origin and mapped the influence of plant part (rhizome versus root), genotype, drying, cutting, and extraction solvent (Peschel et al., 2013). Consistent plant age and growing conditions excluded environmental influences that hamper comparisons of wild plants from different locations. The objective of this study was to investigate the influence of harvest season (3 times a year) and age (3–5 year old plants) across different provenances with the focus on total rosavins (ROStot ) but also cinnamyl alcohol (CA), the aglycon of the three phenylpropenoid glycosides. This compound had only occasionally been considered in previous investigations (Wiedenfeld et al., 2007; Altantsetseg et al., 2007) but is not systematically included in standardisation so far. To exclude potential co-influencing factors we also continued assessing parameters for the production of highvalue starting materials. In order to define and validate sample processing and extraction for the screening (constant factors with potential influence on ROStot and CA) we varied germplasm, plant part, drying conditions, and extraction solvent and also checked individual plant influence (intragenetic variation, plant sex) on the phenylpropenoid content. 2. Material and methods 2.1. Plant and reference material Collected seeds from 9 wild provenances originate from Northwestern European costal areas: Iceland (R2, 50 m above mean sea level (amsl)), Faroe (R7, 30 m amsl), UK (Shetland R3, R9, 10–25 m amsl), from Scandinavia: Finland (R5, 140 m amsl), from the Eastern/Central Alps: Austria (R10, 1750 m amsl), Switzerland (R1, 1500 m amsl), and from the Pyrenees: Spain (R4, 2600 m amsl), France (R8 1800 m amsl) covering roughly the European lowland and mountainous habitats apart from the East (North Russia, Ural) and Southeast (Carpathian mountains). They were authenticated by Dr. Kump (Linz, Austria) and completed with one sample purchased from a commercial seed supplier (R6, Lot: 80012015100 delivery No. 373081 2006, Jelitto, Schwarmstedt, Germany). Voucher specimens have been deposited at the herbarium of the Biologiezentrum der Oberösterreichischen Landesmuseen (Linz, Austria; No: 736649–736656, 779686, 779687). For validation and comparison commercial reference material of different sources were used: 4 herbal drugs (I ‘Rhizomata et radices Rhodiolae rosea’ 50 g cut pieces, Barnaul, Russia; II unlabelled from a market stand 40 g cut pieces, Barnaul, Russia; III ‘R. rosea (rhizoma)’ 50 g cut pieces, Gorno-Altaisk, Russia; IV ‘Rhodiolae radix concisus’ 500 g, Gittelde, Germany) and two products (I R. rosea extract 36.7 g/100 g powder in capsules, Germany); II ‘Arctic root—R. rosea’, powder in capsules, origin UK/sold in Szeged, Hungary. As reference standards salidroside (CAS 10338-51-9), tyrosol (CAS 501-94-0), rosavin (CAS 84954-92-7), and cinnamyl alcohol (CAS 104-54-1, all SIGMA, HU) were diluted in methanol. Stock and derived solutions for HPLC linearity tests were stored at −18 ◦ C.

2.2. Propagation, cultivation, harvest and drying After seeding in early spring 2006, seedlings, re-potted twice, were kept under greenhouse conditions until October. All propagation conditions including peat containing substrate, pot size, water supply on demand, moderate fertilisation in the second half of the first year (Osmocoteplus® NPK 15-10-12) as well as greenhouse conditions were homogenously applied across all provenances. In November 2006, for the main screening 40 individual plants per provenance were planted (4 randomly distributed blocs with 10 individuals each per genotype, 30 cm in and between rows) into an experimental plot at University Reading (51.4419◦ N, 0.9456◦ W, Berkshire, UK, 66 m amsl). For other experiments (plant part, drying, extraction, sample size, plant sex) remaining plants (maximum 16 per provenance) were planted in a neighbouring plot. The cultivation site is characterised by a temperate maritime climate with continental influence (annual precipitation mean 635.4 mm, annual sunshine mean 1522 h) with average high temperature between 23 ◦ C in July/August (maxima 35 ◦ C) and 8 ◦ C in December/January (maxima 15 ◦ C) and average low temperature between 11 ◦ C in July/August (minima 5 ◦ C) and 2 ◦ C in December/January (minima −15 ◦ C). No additional fertilisation or water supply or other cultivation measures apart from weeding were applied. Plants were harvested between March 2009 (cultivation year 3) and November 2011 (cultivation year 5). Plants were manually harvested as a whole, the herb and soil residues removed before rhizome and root were separated and cut (2–6 cm, maximum 1 cm thick) as previously described (Peschel et al., 2013). Root and rhizome fresh weight (FW) and dry weight (DW) were recorded for each plant before and after drying using warm air ventilation at 45 ◦ C for 5 days. Dry samples were stored in paper bags under dark dry conditions at temperatures between 10 and 25 ◦ C. 2.3. Extraction The cut dried drug consisting of 0.5–6 cm long pieces was powdered for 30 s using a grinder (Grindomix GM 200, Germany). After sieving for one minute 5.0 g of the medium fraction (upper limit 0.8 mm mesh diameter, lower limit 0.15 mm) were transferred to 50 mL test tubes and covered with 25.0 mL solvent according to the pharmacopoeial tincture standard to produce a 1:5 ratio between drug and extraction solvent (ethanol 70% v/v, HPLC grade; Molar Chemicals, HU). After moderately shaking for 2 h (Gerhardt, Germany), 5 days maceration at room temperature and another 30 min shaking the tinctures were centrifuged (4500 RPM, 5 min, Rotanta 460, Hettich, Austria) and about 1.6 mL filtered through 0.46 ␮m syringe filter (Labex Ltd., FilterBio® PTFE 13 mm) directly into HPLC vials. Samples were stored at 4 ◦ C in the dark. HPLC analysis was performed within 4 weeks. 2.4. HPLC analysis The direct analysis of tinctures without pre-treatment previously described (Peschel et al., 2013) was slightly modified and is based on the method by Ganzera et al. (2001) without using a phosphate buffer. It allowed for the detection of phenylpropenoids

W. Peschel et al. / Industrial Crops and Products 83 (2016) 787–802

at 254 nm and phenylethanoids at 275 nm without major influence of tannine-caused baseline drifts at relevant retention times. A HPLC Waters 600 Controller, together with a Waters 2707 autosampler and a Waters 2998 PDA detector controlled by EmpowerTM software was equipped with a Luna C18(2) 100A (150 × 4.6 mm, 5 ␮m) column (Phenomenex, UK) thermostated at 40 ◦ C together with a guard column. The mobile phase consisted of water (A), and acetonitrile (B), (HPLC grade) with a gradient elution from 95A/5B in 25 min to 85A/15B and 78A/22B (32 min) at a flow rate of 1.0 mL/min followed by a 8 min wash with methanol (HPLC grade) and an equilibration period for 7 min. Tincture samples prepared as outlined above were injected directly (10 ␮L injection volume). Reference standards salidroside, tyrosol, rosavin, and CA were injected in 6 different concentrations. Every 36 injections the column was washed for 30 min with pure water and 20 min with pure methanol and blanks and a reference standard mix re-applied.

2.5. HPLC assay and validation Salidroside (tR 6.2 (9.2) min, UV ␭max 274 nm) and tyrosol (tR 9.4 min, UV ␭max 274 nm) at 275 nm, as well as rosavin (tR 26.2 min, UV ␭max 248 nm) and CA (tR 31.1 min, UV ␭max 248 nm) at 254 nm (all SIGMA, HU) were quantified by means of external standard. Table 1 shows the limit of detection (LOD) with a signal to noise ratio of 3 or higher and the limit of quantification (LOQ) with a signal to noise ratio of 10 or higher. Additional calculation of the two rosavin-accompanying subordinated phenylpropenoid glycosides rosarin (tR 25.4 min) and rosin (tR 27.1 min both, UV ␭max 248 nm)-using rosavin allowed the summation of the quality indicating marker total rosavins (= ROStot ). We summarised also total phenylpropenoids (PPtot = ROStot + aglycon CA) and total salidroside (SALtot = salidroside plus aglycon tyrosol). Ratios parameters were calculated for some relative comparisons: the rosarin–rosavin–rosin ratio and rosarin–rosavin–rosin–CA ratio (with rosarin set as 1) and the ratio between PPtot and SALtot (with SALtot set as 1), i.e. R. rosea characteristic phenylpropenoids and their aglycon and the characteristic phenylethanoid found in all Rhodiola species plus its aglycon. The precision was checked by repeating measurements of standards at a medium (100 or 500 ␮g/mL) concentration on 5 different days (for r.s.d. values see Table 1). Additionally a reference standard mix (salidroside, tyrosol, CA 50 ␮g/mL, rosavin 125 ␮g/mL) and one extract were injected at 5 different days resulting r.s.d. values of 5.9. and 7.5% (SALtot ), 4.4 and 4.6% (ROStot ), 4.1 and 3.7% (CA), respectively. The linearity was determined using 6 different concentrations per reference standard in the range of 2.5 ␮g/mL to 5 mg/mL with a linear relationship as given in Table 1. Each sample was measured in duplicate.

Constant factors

• Age • Cultivation conditions • Harvest time • Drying • Extraction

(see Sections 2.1–2.3)

Variable factors

• Provenance (3) • Plant part (3)

• R2, R3, R5 • Root, rhizome, herb

n per variable factor Evaluated variables

3 ROStot , CA, SALtot

Statistical analysis

Mean ± s.d., n = 3

Derived variables: PPtot , PPtot :SALtot ratio, rosarin:rosavin:rosin ratio Descriptive only, no mean comparison test for significance

For comparison, four marketed herbal drug samples (Drugs I–IV) were treated in a similar way as well as two products (Prod I, II, see Section 2.1) for which the powder as removed from the capsules was directly used independent from excipients or other ingredients. For each sample 2 macerates were prepared and measured in duplicate. Constant factors

• Extraction

(see Section 2.3)

Variable factors

• Sample origin (6)

• Drugs I–IV • Prod I, II

n per variable factor

2

Evaluated variables

ROStot , CA, SALtot

Statistical analysis

mean ± s.d., n = 2

2 macerates per sample measured in duplicate Derived variables: PPtot , PPtot :SALtot ratio Descriptive only, no mean comparison test for significance

2.6.2. Drying temperature and duration Two 5-year old plants from 3 randomly chosen provenances outside the main screening (R-I, R-II, R-III) were harvested in July and split in rhizome and root. Each of the cut samples (2–6 cm, maximum 1 cm thick) was again divided into two whereof one half of each was dried using warm air ventilation at either 45 ◦ C or 65 ◦ C for 5 days resulting in 4 root samples and 4 rhizome samples at each temperature per genotype. Dry samples were ground (0.8–1.5 mm mesh diameter) and 5 g extracted with 25.0 mL 70% ethanol for 5 days (see above). The content of salidroside, tyrosol (summarised as SALtot ), ROStot , CA and their ratios was determined and expressed in ␮g/mL macerate (Mean ± s.d. of 2 plants 2 samples each, n = 4). Results were pairwise tested for significance (e.g. R-I rhizome ROStot at 45 ◦ C vs. R-I rhizome ROStot at 65 ◦ C) with GraphPad® (InStat 3) statistics software by one-way ANOVA followed by the Bonferroni’s post-test. Significant differences are shown as *P < 0.05. Constant factors

• • • • •

Variable factors

• Drying temperature (2) • (Provenance 3) • (Plant part 2)

• 45 ◦ C, 65 ◦ C • R-I, R-II, R-III • Root, rhizome

n per variable factor

4

2 plants, 2 samples per plant

Evaluated variables Statistical analysis

ROStot , CA, SALtot mean ± s.d., n = 4

2.6. Evaluation of variable factors with influence on the chemical profile—process validation 2.6.1. Plant identity and plant part From 3 authentic provenances (R2, R3, R5) and a previously identified non-authentic provenance (R6) 3-5-year old plants (3 individual plants each), harvested in July, were split into herb, rhizome and root, dried at 45 ◦ C, ground (0.8–1.5 mm mesh diameter) and 5 g extracted with 25.0 mL 70% ethanol for 5 days (see above). The content of salidroside, tyrosol (summarised as SALtot ), ROStot , CA and their ratios was determined and expressed in ␮g/mL macerate (mean ± s.d., n = 3). The chemical profile was further compared using derived variables PPtot , PPtot :SALtot ratio, rosarin:rosavin:rosin ratio (see Section 2.5).

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Age Cultivation conditions Harvest time Drying duration Extraction

(see Sections 2.1–2.3)

One-way ANOVA + Bonferroni post test

Two 4-year old plants from 2 randomly chosen provenances outside the main screening (R-IV, R-V) were harvested in October and split into rhizome and root. Each of the samples was divided

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into two whereof one half was cut into smaller pieces (1–4 cm, maximum 0.5 cm thick) the other in bigger pieces (3–8 cm, maximum 1.5 cm thick). All samples were split into 2 resulting in 4 root samples and 4 rhizome samples for both sizes per genotype. Samples were dried at 20 ◦ C moderate air ventilation until they were completely dry (fine cut samples 10 days, coarse cut samples 30 days). Dry samples were ground (0.8–1.5 mm mesh diameter) and 5 g extracted with 25.0 mL 70% ethanol for 5 days (see above). The content of salidroside, tyrosol (summarised as SALtot ), ROStot , CA and their ratios was determined and expressed in ␮g/mL macerate. Means (± s.d, n = 4) were pairwise tested for significance (e.g. R-IV rhizome ROStot fine cut 10 days vs. R-IV rhizome ROStot coarse cut after 30 days) with GraphPad® (InStat 3) statistics software by one-way ANOVA followed by the Bonferroni’s post-test. Significant differences are shown as *P < 0.05. • Age • Cultivation conditions • Harvest time • Drying temperature (20 ◦ C) • Extraction

(see Sections 2.1–2.3)

Variable factors

• Comminution (size) with subsequent drying duration (2) • (Provenance 2) • (Plant part 2)

• Fine cut 10 days vs. coarse cut 30 days • R-IV, R-V • Root, rhizome

n per variable factor

4

2 plants, 2 samples per plant

Evaluated variables Statistical analysis

ROStot , CA, SALtot Mean ± s.d., n = 4

Constant factors

One-way ANOVA + Bonferroni post test

2.6.3. Extraction solvent and exhaustiveness Influence of solvent polarity: In addition to 70% ethanolic extracts four other extracts were prepared (n = 3) for three drug samples to cover a certain range (rhizome of a four year old plant, rhizome and root of 6 year old plant) using the same procedure but as solvent ethanol 30% v/v, 50% v/v, and 90% v/v (all analytical grade, Molar Chemicals, HU) for comparison of ROStot , CA and SALtot in the macerates (Mean ± s.d, n = 3). Constant factors

Variable factors

n per variable factor Evaluated variables Statistical analysis

• • • •

Provenance Cultivation conditions Harvest time Drying

(see Sections 2.1–2.3)

• Extraction solvent (4) • (Samples 3)

• EtOH 30%, 50%, 70%, 90% • Age 4 rhizome, age 6 rhizome, age 6 root

3 ROStot , CA, SALtot mean ± s.d., n = 3

3 extracts per sample Descriptive only, no mean comparison test for significance

Validation of the procedure: Using two drug samples the sample preparation outlined above (70% ethanol) was repeated twice at two other days to check the consistency of the results. ROStot , CA, SALtot were determined (mean ± s.d., n = 3) and relative standard deviations (r.s.d.) compared to the first extraction. Exhaustiveness of extraction: For three drugs (2 rhizome, 1 root) using 4 different extraction solvents (3 × 30% ethanol, 3 × 50% ethanol, 3 × 70% ethanol, 3 × 90% ethanol) the extraction procedure was repeated additional three times (M2-4) with fresh solvent after filtering and drying the drug sample following the first and each following extraction. ROStot , CA SALtot values (mean ± s.d of mea-

surement in duplicate of 3 samples each) of M1-M4 were compared and expressed as% of the total amount extracted from one sample. Constant factors

• Provenance • Cultivation conditions • Drying

(see Sections 2.1–2.3)

Variable factors

• Extraction repetition (4) • Extraction solvent (4) • Samples (3)

• Macerations M1-4 • EtOH 30%, 50%, 70%, 90% • Age 4 rhizome, age 6 rhizome, age 6 root

n per variable factor 3 Evaluated variables ROStot , CA, SALtot Statistical analysis

mean ± s.d., n = 3

Derived: % per maceration of total amount extracted Descriptive only, no mean comparison test for significance

2.6.4. Intra-genetic variation—sample size requirements Variation of single plants per provenance (seed propagation from one original plant) were tested with 3 year old plants (harvest August) both for composition and yield data. Test 1 (10 individual plants from one provenance): Ten plants from one provenance (n = 10) outside the main screening were harvested, split in rhizome and root, cut, and dried using warm air ventilation at 45 ◦ C for 5 days with determination of FW and DW. Dry rhizome samples were ground, extracted (70% ethanol) and the content of ROStot , CA was determined and expressed in ␮g/mL macerate (see above). Mean (±s.e.m.), minima and maxima, and the relative standard deviation (r.s.d. in%) were determined for n = 10 but also for lower sample sizes (n = 3–6) that were potentially considered for the main experiment: i.e. any 3–6 out of the total 10 for comparison. Significant differences were tested using on-way ANOVA followed by Tukey’s post-test between the mean (n = 10) and means at lower sample sizes (n = 3–6) and also between means at each sample size. Constant factors

• • • • • • •

Variable factors

• Individual plants (10)

n per variable factor

= variable factor

Evaluated variables Statistical analysis

ROStot , CA, DW mean ± s.e.m, r.s.d., n = 10 means ± s.e.m, r.s.d., for n = 6 - n = 2 (any out of 10)

Provenance Age Cultivation conditions Harvest time Drying Plant part (rhizome) Extraction

(see Sections 2.1–2.3)

10 individual plants per provenance One way ANOVA + Tukey’s post test

Test 2 (5 individual plants from 3 provenances): For choosing a sample size for the main experiment resulting in representative random samples per genotype and harvest date, 5 plants each from three provenances (n = 5 for A, B and C) outside the main screening were harvested, processed and analysed as before and mean (±s.e.m), minima and maxima, and the relative standard deviation (r.s.d. in%) were determined for n = 5 but also for lower sample size (n = 3 and n = 4). i.e. any 3 or 4 out of the total 5 for comparison. Significant differences were tested using one-way ANOVA followed by Tukey’s post-test between the minima and maxima of resulting means at lower sample sizes (n = 3–4). Constant factors

• Age • Cultivation conditions • Harvest time • Drying • Plant part (rhizome) • Extraction

(see Sections 2.1–2.3)

W. Peschel et al. / Industrial Crops and Products 83 (2016) 787–802 Variable factors

• Individual plants (5) • (Provenance 3)

n per variable factor

= variable factor

Evaluated variables Statistical analysis

ROStot , CA, DW mean ± s.e.m, r.s.d., min/max, n = 5 mean ± s.e.m, r.s.d., min/max, for n = 4 and n = 3 (any out of 5)

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2.7. Main experiment: influence of plant age and harvest season on composition and biomass 5 individual plants per provenance One way ANOVA + Tukey’s post test

2.6.5. Plant sex Test 1 (4 male and 4 female from 2 provenances): A possible influence of plant sex was tested with 3 year old plants both for composition and yield data. Eight plants each from two provenances (male n = 4, female n = 4, respectively) outside the main screening were harvested, split in rhizome and root, cut, and dried using warm air ventilation at 45 ◦ C for 5 days with determination of FW and DW. Dry rhizome samples were ground and extracted (70% ethanol) and analysed (ROStot , CA) (see above). Mean (±s.d.), were determined for n = 4 and significance tested using ANOVA followed by Tukey’s post test. Constant controlled factors

• Age • Cultivation conditions • Harvest time • Drying • Extraction

(see Sections 2.1–2.3)

Variable factors

• Plant sex (2) • (Provenance 2)

• Female, male • D, E

n per variable factor Evaluated variables Statistical analysis

4 ROStot , CA, DW mean ± s.e.m, r.s.d, n=4

One way ANOVA + Tukey’s post test

Test 2 (retroactive comparison of differences across complete sample matrix): From the main experiment (see 2.7) we compared at 4 harvest dates with records of plant sex (August, November year 3; November year 4, March year 5) the results for male and female plants compared to the total (n = 4 × 9 = 36) across 9 provenances as regards ROStot , CA, (Mean ± s.e.m. from variable n per plant sex). Significant differences were tested using one-way ANOVA (al individual samples, no consideration of provenances) and two-way ANOVA (consideration of provenances) followed by Tukey’s posttest. Additionally ROStot /CA ratios (rhizomes), as well as total DW (root plus rhizome) and the rhizome/root ratio (DW) were compared. Constant factors

• Cultivation conditions • Drying • Extraction

(see Sections 2.1–2.3)

Variable factors

• Plant sex (2) • (Harvest time 4) • (Provenance 9)

• Female, male • Year 3 Aug, year 3 Nov, year 4 Nov, year 5 Mar • Provenance not considered

n per variable factor

Total 36 (9 × 4)

Evaluated variables

ROStot , CA, (DW)

Statistical analysis

mean ± s.e.m, r.s.d, n = 4

n-male and n-female variable per harvest date Derived factors: ROStot –CA ratio, rhizome–root ratio One way ANOVA + Tukey’s post test (no consideration of provenance) Two way ANOVA + Tukey’s post test (consideration of provenance)

Four plants per provenance from 9 provenances (R1–R5, R7–R10) were harvested mid March (start vegetative season before sprouting), mid August (during fructification and start withering of the first generation stems) and beginning November (end of vegetative season) in cultivation year 3, 4, and 5. The herb had to be removed in August (partially in November the wilted herb and occasional 2nd/3rd generation stems) before splitting in rhizome and root, cutting (2–6 cm, maximum 1 cm thick) and drying using warm air ventilation at 45 ◦ C for 5 days with determination of FW and DW. Dry rhizome samples were ground (0.8–1.5 mm mesh diameter) and 5 g extracted with 25.0 mL 70% ethanol for 5 days (see above). The content of ROStot and CA was determined and expressed in ␮g/mL macerate (HPLC) (see above). For the influence of harvest season and age we compared: (1) harvest date means (±s.e.m) and range (minimum, maximum) across all provenances (n = 36) within each year without (oneway ANOVA) and with consideration of provenance differences (two-way ANOVA) and across all years (n = 108) (two-way and three way ANOVA, respectively). (2) annual means (±s.e.m) and range (minimum, maximum) across all provenances (n = 36) as per harvest season without (oneway ANOVA) and with consideration of provenance differences (two-way ANOVA) and across all harvest seasons (n = 108) (two-way and three way ANOVA respectively). In addition we looked at provenance differences as regards composition (ROStot , CA, PPtot , ROStot –CA ratio) as per harvest date (n = 4,one-way ANOVA) and across all seasons and cultivation years (n = 4 × 3 × 3, three-way ANOVA). Constant factors

• • • •

Variable factors (‘treatments’) tested

• Age (3) • Harvest time (3) • (Provenance 9)

n per variable factor Evaluated variables

36 (9 × 4)

Statistical analysis (1) Harvest season comparison within each year with and without consideration provenances (2) Age comparison per harvest season with and without consideration provenances (3) Harvest season comparison across all years with and without consideration provenances (4) Age comparison across all harvest seasons with and without consideration provenances

Cultivation conditions Drying Plant part (rhizome) Extraction

(see Sections 2.1–2.3)

• Year 3–5 • Mar, Aug, Nov • R1–R5, R7–R10

ROStot , CA, (DW)

Derived variables: PPtot , ROStot –CA ratio FW–DW ratio, root–rhizome ratio

(1) Mean ± s.e.m, n = 36 (9 × 4)

(1) One-way and two-way ANOVA + Tukey’s post test

(2) Mean ± s.e.m, n = 36 (9 × 4)

(2) One-way and two-way ANOVA + Tukey’s post test

(3) Mean ± s.e.m, n = 108 (9 × 4 × 3)

(3) Two-way and three-way ANOVA + Tukey’s post test

(4) Mean ± s.e.m, n = 108 (9 × 4 × 3)

(4) Two-way and three-way ANOVA + Tukey’s post test

792 (5) Genotype comparison at each harvest date (9) (6) Genotype comparison (all years and harvest seasons) under consideration of harvest season and age variation

W. Peschel et al. / Industrial Crops and Products 83 (2016) 787–802 (5) Mean ± s.e.m, n = 4

(6) Mean ± s.e.m, n = 36 (3 × 3 × 4)

(5) One-way ANOVA + Tukey’s post test (6) Three-way ANOVA + Tukey’s post test

2.8. Statistical analysis For factor evaluation pre-tests 2.6.1–2.6.3 ROStot , CA and SALtot values (HPLC) represent mean ± s.d. (n = 2–4) according to test design as indicated above and in captions of Figs. 2, 4, 5 and 6. Each single n value is the mean of HMPC measurement in duplicate after validation (2.5). Where useful (n > 3), means were pairwise compared for significance with GraphPad® (InStat 3) statistics software by one-way ANOVA followed by Bonferroni’s post-test. Significant differences are shown as *(p < 0.05). For the cultivation experiment (2.7) and the linked sampling pre-tests (2.6.4 and 2.6.5), ROStot , CA, and DW were calculated as mean ± s.e.m. of all plants per harvest date (n = 36), per year (n = 108) or per season (n = 108) unless otherwise specified (see above). For provenance comparison means ± s.e.m. of 4 individual plants per genotype (at each of the nine harvest dates) and means ± s.e.m of 36 samples per genotype (form all 9 harvest dates) are used. Each single n value is the mean of HPLC measurement in duplicate (2.5). Significance was tested via one-way, two-way or three-way ANOVA plus Tukey’s post-test (R-3.2.1 software) according to analysis as indicated above and specified in captions of Table 3 and Figs. 7, 8 and 9. Significant differences are indicated as *(p < 0.05) or by using different letters when p < 0.05 for provenance comparison. The same test was applied comparing means resulting from different sample sizes and subgroups according to plant sex. Intra-genetic variation between root samples were also expressed as relative standard deviation (±r.s.d.). Correlations between yield and content were tested using Pearson’s correlation test.

Table 2 Exhaustiveness of maceration. Relative percentage of ROStot , CA and SALtot extracted with 1st, 2nd and 3rd maceration (M1-3; M4 always < LoQ) of total amount extracted dependent on the extraction solvent. Mean ± s.d of measurement in duplicate of 3 different drug samples each. ROStot (%)

CA (%)

SALtot (%)

EtOH 30%

M1 M2 M3

60.2 ± 4.8 30.8 ± 1.1 8.9 ± 5.9

50.9 ± 4.0 36.3 ± 3.3 12.8 ± 5.7

51.6 ± 5.4 40.4 ± 2.6 8.0 ± 5.5

EtOH 50%

M1 M2 M3

71.9 ± 9.1 23.7 ± 8.8 4.28 ± 0.3

63.0 ± 1.5 28.0 ± 0.4 9.0 ± 1.9

71.6 ± 9.3 17.9 ± 5.6 10.5 ± 3.7

EtOH 70%

M1 M2 M3

77.5 ± 8.3 17.1 ± 6.5 5.3 ± 2.9

73.3 ± 9.7 23.5 ± 8.3 3.1 ± 1.5

62.0 ± 12.7 23.7 ± 10.6 14.3 ± 8.1

EtOH 90%

M1 M2 M3

80.5 ± 7.7 14.6 ± 5.6 4.8 ± 2.4

85.9 ± 5.0 11.6 ± 3.7 2.5 ± 1.3

62.8 ± 12.6 25.8 ± 9.5 11.4 ± 5.1

3. Results 3.1. Screening validation and robustness – factor evaluation 3.1.1. Germplasm authenticity We compared profiles of rhizome, root and herb of three previously authenticated R. rosea samples (same age and harvest) and a deviating commercial accession, a hybrid of R. rosea with another Rhodiola species with distinct morphological characteristics (Peschel et al., 2013) (Figs. 1 and 2). Extracts from the three authentic rhizome samples contained 0.7–1.3 mg/mL ROStot , 0.1–0.3 mg/mL CA and 0.1–0.6 mg/mL SALtot ; the non-confirmed accession in contrast 0.17, 0.02 and 0.18 mg/mL, respectively. As absolute values may differ according to multiple factors, for identification the ratio between characteristic phenylpropenoids and phenylethanoids may be useful with PPtot clearly higher than SALtot . For authentic rhizomes samples here it was 2.5–7.0:1 but only 1.1:1 for the non-authentic sample. The difference between rhizome and root for rosavins (2–3 fold lower root values) is less pronounced for CA and was not systematically observed for SALtot . The overground parts contained no detectable rosavins

Fig. 1. HPLC chromatogram (␭ = 254 nm) of 70% EtOH extracts. (A) rhizome and (B) root of an authentic compared to (C) rhizome and (D) root of a non-authentic R. rosea accession cultivated in South England (harvest July year 5) with characteristic phenylpropenoids rosarin (1), rosavin (2), rosin (3), cinnamyl alcohol (CA) (4).

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Fig. 2. Total rosavins (ROStot ), cinnamyl alcohol (CA) and total salidroside (SALtot ) in rhizome and root of 3 authentic provenances and a non-confirmed accession (R6). (70% EtOH extracts of 5-year old plants; all ␮g/mL Mean ± s.d. of 3 plants each).

Fig. 3. HPLC chromatogram (␭ = 254 nm) from four marketed herbal drugs (A–D) and 2 products (E + F) indicating authentic R. rosea for A–C and E, absence of R. rosea (D) or low proportion of R. rosea (F): rosarin (1), rosavin (2), rosin (3), CA (4).

and very low amounts of CA and SALtot (<35 ␮g/mL, data not shown). Although differences between rhizome and root samples varied between plants the consistently higher rhizome content suggested rhizome analysis only for a major screening—also to avoid variations due to distinctive rhizome/root ratios between samples. Rosavin was always the predominant phenylpropenoid across all samples, yet the rosarin:rosavin:rosin ratio also shows some differences between authentic (rhizome 1:2.8–3.2:0.3–0.4, root

1:1.7–3.5:0.3–0.5) and non-authentic samples (rhizome 1:6.8:0.4, root 1:6.8:0.5). Three marketed drug samples originating from Russia, one traded in Germany and 2 products sold in Austria and Hungary were tested for comparison. They revealed similar features for 3 drugs and 1 product (PPtot –SALtot ratio 2.8–4.2:1) but for one drug and one product unlikely R. rosea as (only) source plant (Figs. 3 and 4). One drug sample (labelled Rhodiolae radix concisus) had a ROStot below LoD indicating absence of R. rosea while one product had

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W. Peschel et al. / Industrial Crops and Products 83 (2016) 787–802

Fig. 4. ROStot , CA and SALtot in four marketed drugs and two products (Mean ± s.d. of 2 samples each measured in duplicate).

very low ROStot values compared to high salidroside (PPtot –SALtot ratio 0.05:1) indicating a mixture of authentic R. rosea with possibly other Rhodiola species not containing rosavins. 3.1.2. Drying temperature and duration We found no significant ROStot differences between samples dried at 45 or 65 ◦ C for 5 days (Fig. 5A for rhizomes) apart from a slight trend that rosavins may decrease at higher temperature. SALtot values in contrast partially increased at higher temperatures, while no clear influence of the drying temperature on CA could be observed. In a test on extended drying duration (fine cut versus coarse cut at 20 ◦ C), both ROStot and SALtot were found reduced by 5–25% and 10–45%, respectively, when slowly dried (30 days) compared to 10 days (Fig. 5B). The bigger pieces kept over 2 weeks a gum like texture with only the outer parts dry. For the screening, all samples were fine cut and dried at 45 ◦ C ± 10 ◦ C for 6 days using the same drying equipment. Minor deviations in temperature and duration are not expected to influence ROStot and CA values. 3.1.3. Extraction solvent Rosavins are best extracted with 70–90% ethanol, while CA was found highest in 50–70% ethanolic extracts (Fig. 6). When the same sample was powdered and extracted at three different days, variations were between 7.7% and 6.7% (r.s.d). The extent of extraction exhaustiveness depended on extraction solvent. Under our conditions used for the screening (5 days maceration, rhizome, 70% ethanol) the first extraction resulted in average 77.5% (CA 73%) of the total extractable amount, the second in 17.1% (CA 23.5%), the third in 5.3% (CA 3.1%) with all s.d. below 10% (Table 2). In the fourth the amounts were below LoQ in all cases. While for quality control a triple maceration would be advisable to determine the absolute total amount of phenylpropenoids contained in the drug, for screening purposes the single maceration, yielding consistently about 75% both for ROStot and CA showed to be suitable to compare samples via 70% ethanolic extracts. In addition, from this yield and the drug solvent ratio (1:5) total amounts in the drug can be estimated (% w/w rosavins in DW drug = x mg/mL × 1.25 × 5 mL/g/10). For instance a tincture containing 0.5 (1.5, 2.5) mg/mL total rosavins corresponds to about 0.31 (0.94, 1.56, respectively) % in the dry drug. 3.1.4. Intra-genetic variation in content and biomass When sampling simultaneously 10 single plants from one provenance (August 2009) the ‘intra-genetic’ (seed propagation) variation for ROStot (rhizome, 70% EtOH; range 1385–1970 ␮g/mL,

r.s.d. 11.7%) was more consistent than for CA (rhizome 70% EtOH; range 164–374, r.s.d. 33.9%) but also yield (total DW; range 71.6–171.5 g, r.s.d. 30.0%). No correlation or inverse correlation between yield and phenylpropenoids (CA or ROStot ) was detected. No significant differences between the total mean (n = 10) and means for reduced sample sizes down to n = 3 (any 3 out of 10) were detected. However, differences between the means from reduced sample sizes were partially significant for n = 3 (ROStot ) and n = 4 (CA) which would compromise comparison of provenances/. We therefore checked for 3 provenances 5 plants (means, range and r.s.d. for n = 5) and compared the results with n = 4 (any of the 5) and n = 3 (any of the 5) (Suppl. data Table 1). Also here, variation in CA and biomass was higher than for ROStot . While for n = 4 no significant differences between resulting means were detected (one exception for CA) at sample size n = 3 deviations to the mean (n = 5) became more substantial. Significantly differing random samples means would not allow considering them representative for comparison. Consequently, as compromise between precision and acceptable expenditure the screening was performed with 4 samples per provenance and harvest date. 3.1.5. Plant sex No significant differences were detected between samples from male (n = 4) and female (n = 4) representatives of 3-year old plants tested for 2 provenances at one harvest date (A: ROStot ♂ 1695 ± 207 ␮g/mL, ♀ 1611 ± 245 ␮g/mL; CA ♂ 195 ± 64 ␮g/mL, ♀ 234 ± 77 ␮g/mL; total DW ♂ 129 ± 37 g, ♀ 103 ± 34 g) and (B: ROStot ♂ 1336 ± 227 ␮g/mL, ♀ 1289 ± 375 ␮g/mL; CA ♂ 180 ± 59 ␮g/mL, ♀ 204 ± 62 ␮g/mL; total DW ♂ 82 ± 26 g, ♀ 90 ± 31 g). Subsequently, in the following main screening the plant sex was not considered when taking randomly n = 4 samples per harvest date and provenance. We recorded, however, plant sex at four different harvest dates and retroactively compared across all provenances male and female plants (Suppl. data Table 2). Again, no significant differences were detected with usually more male than female samples. A trend that male plants develop more biomass and have also slightly higher ROStot content might be indicated, however, cannot be stated from the data here across 9 different provenances and would require a higher sample size from one genotype. 3.2. Phenylpropenoid content of R. rosea rhizomes according to harvest season and age Across all samples and provenances the ROStot content in rhizomes was substantially higher when harvested in March than in August or November alongside a decrease from year 3 to year 5 under our cultivation conditions. Differences between harvest dates within each year where mostly significant, both with and without consideration of the factor provenance. Differences between years were all significant when comparing each harvest date separately (Table 3). Equally harvest season differences across all years and age differences across all harvest seasons where mostly highly significant with and without consideration of the provenance influence (Fig. 7A and C). CA appears equally determined by the harvest season and plant age but to less extent. The March harvest produced always significantly higher CA values compared to August or November; while the latter differed not significantly in cultivation year 4 and 5 (Table 3). Age differences were not always significant, even under consideration of provenance variation, when comparing harvest seasons to each other. When comparing harvest seasons across all years and age differences across all harvest seasons, ‘March CA values’ surpassed significantly those obtained at later harvest dates and ‘year 3 CA values’ those from year 4 and 5, respectively (Fig. 7B and D).

W. Peschel et al. / Industrial Crops and Products 83 (2016) 787–802

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Fig. 5. ROStot , CA and SALtot according to drying conditions. Mean ± s.d. (n = 4). (A) 45 ◦ C versus 65 ◦ C (rhizomes, 5 days), (B) fine cut (10 days) versus coarse cut (30 days) (rhizome or root, all at 20 ◦ C). Sicnificant differences (p < 0.05, one-way ANOVA with Bonferroni’s post test) between treatments (temperature or cut) per sample are indicated with *.

The derived ROStot /CA ratio remained consistent between 7 and 10 despite a wide range. Consequently when glycosides (ROStot ) and aglycon (CA) are summarised as the total phenylpropenoid content, this PPtot value reflected largely the ROStot pattern with about 10% higher values (Table 3). It decreased from March cultivation year 3 (average 3.7 mg/mL) to November cultivation year 5 (average 1.0 mg/mL) corresponding to 2.3 % and 0.6 % (m/m) in the dry rhizome, respectively. All 9 provenances followed a similar ROStot pattern regarding harvest season (maximum in March) and age (maximum in year 3) with partially significant differences (Fig. 8). The influence of genetically determined potential and adaptation to a temperate climate altered with cultivation duration and annual influence. The order was not consistent as per harvest date, season or year not allowing unambiguously ascribing consistent high values to single provenances. At the beginning of year 3 a generally high phenypropenoid

content was found while this deteriorated the next two years with particularly hot and dry summers. Overall, the age and season influence appears higher than the genetic one from the spectrum tested in this study. When comparing across all harvest seasons and years under adjustment of those differences (three-way ANOVA) three provenances (R1, R4, R7) exhibited a slightly, partially significant superior ROStot and PPtot content, while one provenance (R9) contained significantly less phenylpropenoids (Fig. 9A and C). The CA content per provenance followed largely the overall pattern (higher in spring and year 3) (Suppl. data Fig. 1). However, in contrast to the ROStot content we noted more profound and consistent genotype differences (R1, R4, R8 and R10 lower than the other five) independent from season and age. This was confirmed when calculated and adjusted over all years and seasons with significant differences in the CA content and more obvious significant demarcation of the ROStot -CA ratio (Fig. 9B and D). Three groups can be

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W. Peschel et al. / Industrial Crops and Products 83 (2016) 787–802

Fig. 6. ROStot , CA and SALtot in macerates from two R. rosea rhizome samples according to extraction solvent (Mean ± s.d, n = 3).

distinguished: R2, R3, R5, R7 and R9 (all ‘Northern’ origin: Iceland, Shetland, Faroe, Finland) had ratios in the range between 4 and 8 (single plant range 2–17), R1 and R10 (Alpine origin: Switzerland and Austria) 13–16 (range 6–27) and R4 and R8 (Pyrenean origin: France and Spain) 18 and 21 (range 9–32). A possible genetic determination of the relative ROStot -CA ratio vis-à-vis the more season-determined absolute ROStot , PPtot or CA values is illustrated in Fig. 10. Independent from season and age, the phenylpropenoid spectrum i.e. the relative ratio between the three rosavins and their aglycon (rosarin–rosavin–rosin–CA proportions) was quite consistent within each provenance (Fig. 11).

3.3. Yield and biomass versus content The yield development over 3 years without fertilisation is shown in Table 4. The overall growth from average 56 g (first samples March 2009, range 19–103 g) to 99 g (samples November 2011, range 25–237 g) DW per plant is not representative, because in September year 3 all plants had to be transferred to a new experimental plot. The subsequent growth inhibition is visible in the average data from November 2009 onwards. Overall rhizome surpassed root biomass yielding about double the dry weight (rhizome-root ratios approximately 1:0.5). A seasonal influence

Table 3 ROStot , CA, (all in ␮g/mL) and derived variables ROStot /CA ratio and PPtot (␮g/mL) in 70% EtOH extracts from rhizomes of 3–5 year old R. rosea cultivated in South England (mean ± s.e.m and range of 36 samples per harvest date across 9 provenances).

ROStot

CA

ROStot - CA ratio x:1

Range

PPtot

Mean

Range

Mean

Range

Mean

(±s.e.m)

(min–max)

(±s.e.m.)

(min–max)

3320

2399 - 4424

372

77 - 664

8.9

4.6 - 29.0

3692

2903 - 4951

69 - 521

8.1

2.2 - 28.6

2116

1114 - 2942

56 - 493

7.2

2.6 - 22.0

1452

671 - 2551

96 - 412

9.7

4.0 - 27.7

2654

1521 - 3368

19 - 389

9.0

4.2 - 29.5

1841

995 - 2768

15 - 405

7.9

2.6 - 27.9

1667

631 - 2851

623 - 446

9.1

4.0 - 29.4

2248

1335 - 3181

31 - 408

7.9

2.8 - 31.8

1456

682 - 2630

23 - 355

7.3

2.3 - 28.7

990

570 - 1511

(min–max)

Range (min–max)

Year 3 Mar

*

951 - 2736

233

597 - 2334

*

1275

(±19.5)

( )

*

*

*

(±84.5)

*

1883

Nov

(±31.4)

*

Aug

*

(±76)

177

*

( ) *

*

( ) *

(±80.8)

(±20.6)

Year 4 2407

1257 - 3204

*

185

*

*

782 - 2485

( )

*

Nov

247 (±19.4)

*

1656

*

Aug

*

(±81.4)

*

*

*

( )

Mar

(±73.1) 1481

455 - 2674

(±15.2) 186

*

(±95.9)

(±16.6)

Year 5 1070 - 2928

*

565 - 2413

Nov

855 (±36.8)

163 (±17.4)

*

(±72.1)

*

*

1292

224 (±15.4)

*

(±80.8) Aug

*

2024

( )

*

Mar

499 - 1454

137 (±16.6)

*Mark significance p < 0.05 between ROStot or CA according to one-way ANOVA (no consideration of provenance). (*) Mark significance p < 0.05 between ROStot or CA according to two-way ANOVA (consideration of provenance).

W. Peschel et al. / Industrial Crops and Products 83 (2016) 787–802

A

B ooo

4000

ooo

400

***

ooo

***

***

***

3000

300 CA (µg/mL)

ROStot (µg/mL)

ooo

ooo

*** 2000

1000

200

100

0

0 Aug

Mar

Nov

Aug

Mar

C

Nov

D ooo

4000

ooo

***

400

***

ooo

ooo

**

** 3000

300 ooo

CA (µg/mL)

ROStot (µg/mL)

797

*** 2000

1000

200

100

0

0 year 3

year 4

year 5

year 3

year 4

year 5

Fig. 7. ROStot and CA in rhizomes of R. rosea harvested in March, August, November of cultivation years 3; (70% EtOH extracts, mean ± s.e.m. n = 108). (A + B) Comparison of harvest seasons (mean all years); (C + D) Comparison of age (mean all harvest seasons). Significance (one-way ANOVA with Tukeys post test) without consideration of provenance is indicated as *, ** and *** (p < 0.05, p < 0.01; p < 0.005). Significance (two-way ANOVA with Tukey’s post test) with consideration of provenance differences (9 × n = 12) is indicated as o , oo and ooo (p < 0.05, p < 0.01; p < 0.005).

as regards water content of root and rhizome is indicated by the loss on drying with FW-DW ratios of 3.5–3.6:1 when harvested in August compared to 4.0–5.1:1 in March and 3.8–4.1:1 in November. Differences between provenances were less pronounced in year 3 but diversified substantially in year 4 and 5. Three genotypes (R1, R5, R8) seemed to cope best with growing conditions not found in natural habitats of the species.

Due to the sub-optimum conditions (climate, nutrition) but mainly the transfer and subsequent growth depression, the yield was not considered representative for statistical analysis or provenance comparison. Nonetheless we checked to which extent the phenylpropenoid content relates to the biomass. We found no correlation between the ROStot , the CA or the PPtot content against the biomass for the whole sample matrix, as per harvest date (all

Table 4 Yield (DW), fresh-dry weight ratio and rhizome-root ratio (DW) of 3–5 year old samples of R. rosea cultivated in South England (mean ± s.e.m., range of 36 samples per harvest date across 9 provenances). Total DW (g/plant)

FW-DW ratio (x:1)

Rhizome/root ratio (1:x)

Mean (±s.e.m.)

Range (min–max)

Mean (±s.e.m.)

Range (min–max)

Mean (±s.e.m.)

Range (min–max)

55.9 (±4.3) 68.8 (±4.2) 59.4 (±4.3)

18.5–103.1

3.96 (±0.35) 3.53 (±0.37) 3.72 (±0.29)

3.49–4.54

0.62 (±0.19) 0.47 (±0.20) 0.44 (±0.23)

0.34–0.81

32.8–68.1

Nov

72.7 (±4.5)

39.7–112.7

0.31 (±0.15) 0.46 (±0.39) 0.47 (±0.31)

0.16–0.52

29.7–98.5

4.26 (±0.32) 3.59 (±0.45) 4.09 (±0.66)

3.83–4.59

Aug

56.6 (±2.2) 65.3 (±2.5)

73.7 (±9.7) 85.0 (±5.8) 99.1 (±9.6)

26.6–100.8

5.07 (±0.79) 3.53 (±0.28) 3.76 (±0.25)

3.93–6.44

0.52 (±0.21) 0.48 (±0.15) 0.48 (±0.30)

0.21–0.81

Year 3 Mar Aug Nov Year 4 Mar

Year 5 Mar Aug Nov

31.2–119.4 16.5–112.2

31.3–135.8 24.9–236.6

3.00–4.56 3.31–4.33

3.19–4.27 2.69–5.17

3.14–4.04 3.40–4.12

0.22–0.93 0.21–1.07

0.24–0.69 0.11–1.03

0.26–0.67 0.13–1.03

798

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Fig. 8. ROStot (70% EtOH extracts, mean ± s.e.m., n = 4) in 3–5 year old rhizomes from 9 provenances of R. rosea cultivated in South England and harvested in March, August or November. Significance between provenances harvested at the same date (one-way ANOVA plus Tukey’s post test) is indicated using different letters when p < 0.05.

clones), or as per clone (per harvest season all 3 years). Across all samples the highest-content samples (lower biomass) were harvested in spring year 3, vice versa highest yield (substantially decreased content) in November year 5 under our conditions (Fig. 12A). Depending on the specific goal of the cultivation a compromise between content (e.g. according to pharmacopoeial specification) and yield (i.e. cultivation efficiency for the grower) may be achievable in August after 3 years cultivation or March year 4 or 5. Different provenance may be suitable for short term cultivation (high ROStot using fast growing clones delivering a reasonable yield after 3 years already) or longer cultivation periods (higher yields compromising on content using clones still being within pharmacopoeial specification) (Fig. 12B and C). As minimum threshold a biomass of 50 g DW per plant and the current requirement in the USP (ROStot 0.3% DW corresponding to approximately 0.5 mg/mL in our extracts) may be chosen. Under our conditions a focus on the highest content would favour three provenances harvested in March year 3 (Fig. 12B). For the opposite benchmark, i.e. harvest at starting decay of rhizomes in August year 5 (focus on maximum yield) favours four provenances that are likely to deliver herbal drugs according to pharmacopoeial minimum requirements (Fig. 12C).

4. Discussion 4.1. Influence of age and harvest season on the phenylpropenoid content in cultivated R. rosea We showed for the first time systematically that the harvest season has a major influence on the rosavins content when cultivated in a temperate climate. Few previous reports mention seasonal influences. Buchwald et al. (2006) reported 2-3 peaks during the vegetation period in ‘root’ from cultivation in Poland (year 4: peak end July and early October; year 5: end of June, end of August, mid November). The authors linked it to aboveground parts development and postulated overall a high content with ‘first stem generation development’ and during fructification and lower content during the second generation development (August, September). Platikanov and Evstateva (2008) equally refer to shoot generations (more detailed described by Przybyl et al., 2008) when reporting salidroside variations in roots and rhizomes cultivated at higher altitude in Bulgaria and harvested at start of the vegetation period, during flowering, blossoming and the end of vegetation period with some increase in 2–3 year old male rhizomes. NMRbased metabolomic analysis indicated also a decrease of rosavins content at the end of seasons with samples taken May to September

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Fig. 9. ROStot , CA, PPtot and ROStot /CA ratios (70% EtOH extracts, mean ± s.e.m., n = 36) in 3–5 year old rhizomes from 9 provenances of R. rosea cultivated in South England and harvested in March, August or November. Significance between provenances (three-way ANOVA plus Tukey’s post test) is indicated using different letters when p < 0.05.

A

B

450

30

ROStot/CA ratio x:1

CA (µg/mL)

R2 R5R3 R7

300 R9 150

R1

R10

R4 R8

R8

25

R4

20 15 10 R9

5

0

R1

R10 R3

R5

R7 R2

0 0

1000

2000

3000

ROStot (µg/mL)

0

1000

2000

3000

PPtot (µg/mL)

Fig. 10. (A) CA versus ROStot , and (B) ROStot /CA ratios versus PPtot (70% EtOH extracts, mean ± s.e.m., n = 36) in 3–5 year old rhizomes from 9 provenances of R. rosea cultivated in South England and harvested in March, August or November. Significantly different groups (across all seasons and years) are circled (CA in A), (ROSand tot /CA ratio in B).

in wild populations of the Swiss Alps which was not the case for salidroside (Ndjoko Ioset et al., 2011). The usual harvest of root drugs or foods at the end of the vegetative season is followed by most producers and investigations but may not be the optimum for R. rosea. Instead harvest in spring before or at sprouting is indicated. This might be supported by data that the rosavin content of the vegetative buds is rather high (comparable to the rhizome according to Kołodziej and Sugier, 2013). Despite lower ROStot content some practical advantages were linked in our experiment with an ‘end of summer’ harvest (yield increase, less expenditure for harvesting, cleaning, drying). The substantial decrease year 3–5 was unexpected but cannot be generalised due to cultivation probably on the edge of what is possible in terms of temperature and humidity. Some genotypes were better able to adapt to warmer conditions than found in their natural habitats. Annual ROStot differences were reported between the same wild populations (e.g. Malnoe et al., 2009 for 2 following years, Swiss alps, age unknown) but also cultivated plants: year 4 > year 5 during

cultivation in Poland (Buchwald et al., 2006), year 3 > year 4 during cultivation in Finland (Galambosi et al., 2009), relatively high in year 1, lower in year 2/3, peak in year 5 (Przybyl et al., 2008, Poland), and more recently also from 7 years cultivation in Poland a peak in year 4 and 5 (Kołodziej and Sugier, 2013). Most authors report also the starting decay of underground parts from year 5 onwards changing rhizomes/roots ratios, yield, drying expenditure (rotten parts often soaked with moisture) and content. Such plant ageing seems to start much earlier during cultivation than in the wild under more harsh conditions—presumably depending on the length of the vegetation period and subsequent generations of shoots (Przybyl et al., 2008; Galambosi et al., 2009). For the CA content, no systematic investigation of season influence is published so far. We found values at about 5–30% of the ROStot content and roughly in line with rosavins in terms of seasonal profile and age under our conditions. This went in parallel with a presumably genetic determination: a lower CA proportion for central and south European provenances (Alps, Pyrenees) and a higher for those originating from Iceland, Faroe, Shetland and Finland.

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Fig. 11. Relative phenylpropenoid profile (%, mean of 4 plants per harvest date) for one representative of each group with significant ROStot –CA ratio differences: (A) R2-Iceland, (B) R1-Alps, (C) R4-Pyrenees.

Because of the limited number of geographic representatives in this study, such determination needs more data for confirmation. In conclusion, a drug highest in ROStot and CA is in temperate conditions ideally harvested in early spring. 4.2. Other factors influencing the phenylpropenoid content in R. rosea derived preparations 4.2.1. Genotype From 10 genotypes originally included in this study one (R6) had to be excluded as a possible hybrid of R. rosea with another species indicated by obvious divergent morphological and organoleptic characteristics of the plant (Peschel et al., 2013). In contrast, the dry herbal drug was difficult to distinguish, which might be the reason why it is easy to replace authentic material in cut and powdered form. Because the phenylpropenoid content (both, ROStot and CA) is absolute considerably age/harvest dependent, inferiority due to species substitution was best signalled by the relative PPtot -SALtot ratio (>2.5:1 for rhizomes from authentic R. rosea).

Fig. 12. (A) PPtot (␮g/mL, 70% EtOH extracts from rhizomes) in relation to biomass (rhizome + root DW) of 3–5 year old R. rosea cultivated in South England (mean of 36 samples per harvest date across 9 provenances) harvested in March, August or November. (B + C) ROStot (␮g/mL, 70% EtOH extracts) in rhizomes of R. rosea. from 9 provenances (n = 4) versus rhizome DW (g) when harvested (B) March year 3 and (C) August year 5. The horizontal dashed line marks the minimum requirement according to USP (0.3% DW in the drug corresponding to 500 ␮g/mL in the extract); the vertical dashed line a minimum DW of 50 g per plant considered economically justified.

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The same applied to one of four traded drugs and one of two randomly chosen products. The examples show that wrongly labelled seeds, herbal drugs and finished products can currently reach the European market, if no HPLC profiles, identity tests and key marker determination are applied. It confirms previous reports by Ma et al. (2011) and is already addressed in test requirements such as the Australian R. rosea extract standard to distinguish from R. crenulata (Hook. f. & Thomson) H. Ohba and R. sacra (Prain ex Raym.-Hamet) S.H. Fu (TGA 2014). With one exception, particularly salidrosiderich genotypes in the Swiss Alps described by Malnoe et al., 2009; a minimum two-fold higher ROStot versus salidroside content is found in all literature from authentic material independently from rhizome or rhizome + root analysis and solvents used. From the 9 tested authentic provenances a genetic determination of the phenylpropenoid content can be stated and significant differences can be found, however, we did not find a consistently superior clone or provenance group. It can be assumed that all tested genotypes are location-and climate-dependent suitable for cultivation considering content, growth and robustness. The influence of harvest season, ages and plant part was more relevant than provenance although plants originate from very different locations in Europe. Conclusions on superior types from specific regions cannot be made on the basis of this limited sample range. 4.2.2. Plant part Our previous report that rhizomes contain significantly more rosavins than roots (about 1.5–3 times more) has been confirmed also for CA (about half the content in roots). We additionally analysed also the herb in its freshest most abundant status (July, ‘first stem generation during fructification), and found minor amounts of SALtot and CA but rosavins below the detection limit. A previous report gives considerable ROStot values for ‘overground parts’ (about half of rhizome values) and ‘tips’ (similar or even higher than rhizome values) (Kołodziej and Sugier, 2013). However, as not defined and only documented via a picture it is assumed that the ROStot content derives mainly from rhizome parts. As R. rosea is often sold as ‘root’ without distinction between rhizoma and radix but usually a mixture of both, different proportions of the two plant parts are likely to contribute to quality issues and the wide range of the ROStot content found and reported (Peschel et al., 2013). 4.2.3. Drying A 20 ◦ C difference in drying temperature (45 ◦ C versus 65 ◦ C) did not significantly influence the phenylpropenoid content with a comparable drying duration. However, when dried at low temperature (18 ◦ C) it is indicated to cut the drug into smaller pieces as otherwise a significantly prolonged drying time may not only pose a risk for microbial contamination (moist inner parts) but also negatively influence the ROStot content. 4.2.4. Solvent We found optimum extraction of rosavins with 70–90% ethanol with a 75–85% yield with the first maceration, while 30–50% ethanolic extracts contain significantly less rosavins. It can be assumed that traditional ethanolic tinctures have been prepared with a range of alcohol strengths, yet the resulting ‘dosage’ in phenylpropenoids – absolute and in relation to other compoundsis substantially influenced by the choice of solvent. Therefore specification and standardisation is required as otherwise use and effects of one R. rosea preparation is not transferable to others. 4.2.5. Plant sex We did not find consistent significant phenylpropenoid differences according to plant sex. In view of heterogeneous findings in this respect (Galambosi et al., 2009; Malnoe et al., 2009), the slow

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growth, the still prevailing seed propagation, and several more relevant influencing factors it may not be economically justified trying to produce male or female plants only. It may be possible for a targeted wild collection during the vegetative period. However, for conventional cultivation, only after optimisation of other production factors it might be worthwhile to consider the cultivation of e.g. only male plants once a stable high-value cultivar is selected and vegetatively propagated. 4.3. Profile and suitability of rhizomes derived from R. rosea cultivated under temperate conditions The ROStot minimum requirement by the USP monograph is 0.3% (DW). With our results of 0.31–2.6% for the dry rhizome all drug samples would comply. However, assuming a usual mix between root and rhizome the samples from some genotypes when harvested in November could fail minimum requirements. The range of more recently reported ROStot is 0.2–3.7% DW for rhizome/root mixtures (or not specified plant part), 0.4–5.5% for rhizomes and 0.5–3.3 for roots (Altantsetseg et al., 2007; Ampong-Nyarko et al., 2011; Buchwald et al., 2006; Galambosi, 2006; Galambosi et al., 2007, 2009; Kołodziej and Sugier, 2013; Malnoe et al., 2009; Przybyl et al., 2008; Wiedenfeld et al., 2007). CA is so far not considered for R. rosea quality control (such as identity tests or minimum amounts) although usually detected. It constituted here up to 20% of the PPtot content. CA is most known from its occurrence in cinnamon species and use in fragrances, has sensitising properties but occasionally other own pharmacological effects including anti-inflammatory and vasodilatory activities are described (Kang et al., 2012). The role of CA in rosavins biosynthesis has been investigated and it has been used in attempts to increase rosavins in root cultures of Rhodiola species (Grech-Baran et al., 2014). However little is known on stability, pharmacokinetics and metabolism of phenylpropenoid glycosides and the rate of CA release during storage and oral use of R. rosea drugs and extracts. Previously CA contents have been reported for R. rosea and differ considerably according to plant origin, plant part, extraction solvent and analysis: 0.012–0.83% DW for rhizome/root mixtures (or not specified plant part), 0.03–0.05% for rhizomes and about 0.02% for roots (Dascaliuc et al., 2008; Altantsetseg et al., 2007; Galambosi et al., 2007; Galambosi et al., 2009; Malnoe et al., 2009; Przybyl et al., 2008; Wiedenfeld et al., 2007). Given that several of these studies were not performed with the solvent or plant part optimum it is not surprising that our results (rhizome, EtOH 70%) of 25–602 ␮g/mL (corresponding to about 0.015–0.37% DW) are mostly found in the upper part of the previously reported CA spectrum. The possible genetic determination of the ROStot /CA proportion as indicated from our results, suggests that CA may not only be an artefact due to hydrolysis in the dried drug or derived extracts. From a quality perspective it could be used as additional marker for identity and eventually specification and stability. From the pharmacological perspective it might be useful to add to the rosavin content and summarise total phenylpropenoids as long as the active principle and mode of action of R. rosea in different indications are not known and phenylpropenoids as a whole considered as potentially contributing to the activity (Kurkin, 2013). Acknowledgements We gratefully acknowledge the support of Elisabeth Williamson (The School of Pharmacy University Reading, UK), Wolfgang Kainz (AGES, Dept. Pflanzengenetische Ressourcen, Linz, Austria), Martin Pfosser (Botanische Arbeitsgemeinschaft am Oberosterreichischen Landesmuseum, Biocenter, Linz, Austria), Anne Starker (Berlin,

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Germany) and Aidan Slingsby (London, UK) without whom collection, cultivation, sampling and processing would not have been possible. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.indcrop.2015.10. 037. References Altantsetseg, K., Przybył, J.L., Zenon, W., Geszprych, A., 2007. Content of biologically active compounds in roseroot (Rhodiola sp.) raw material of different derivation. Herba Polon. 53, 20–26. Ampong-Nyarko, K., Lutz, S., Sloley, B.D., Piquette, K., Zhang, Z., 2011. Assessment of soil productivity for roseroot (Rhodiola rosea L.) cultivation in Alberta. Z. Arzn. Gew. Pfl. 16, 156–162. Buchwald, W., M´scisz, A., Krajewska-Patan, A., Furmanowa, M., Mielcarek, S., Mrozikiewicz, P.M., 2006. Contents of biologically active compounds in Rhodiola rosea roots during the vegetation period. Herba Polon. 52 (4), 39–43. Dascaliuc, A., Calugaru-Spatatu, T., Ciocarlan, A., Costica, M., Costica, N., Krajewska-Patan, A., Dreger, M., M´scisz, A., Furmanova, M., Mrozikiewicz, P.M., 2008. Chemical composition of golden root (Rhodiola rosea L.) rhizomes of Carpathian origin. Herba Polon. 54 (4), 17–27. Galambosi, B., 2006. Demand and availability of Rhodiola rosea L. raw material. In: Bogers, R.J., Craker, L.E., Lange, D. (Eds.), Medicinal and Aromatic Plants. Springer, pp. 223–236 (Chapter 16). Galambosi, B., Galambosi, Z., Slacanin, I., 2007. Comparison of natural and cultivated roseroot (Rhodiola rosea L.) roots in Finland. Z. Arzn. Gew. Pfl. 12, 141–147. Galambosi, B., Galambosi, Z., Uusitalo, M., Heinonen, A., 2009. Effects of plant sex on the biomass production and secondary metabolites in roseroot (Rhodiola rosea L.) from the aspect of cultivation. Z. Arzn. Gew Pfl. 14, 114–121. Ganzera, M., Yayla, Y., Khan, I.A., 2001. Analysis of the marker compounds of Rhodiola rosea L. (Golden Root) by reversed phase high performance liquid chromatography. Chem. Pharm. Bull. 49, 465–467.

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