SUPPLEMENTARY MATERIAL Differential response of terpenes and ...
SUPPLEMENTARY MATERIAL Differential response of terpenes and anthraquinones derivatives in Rumex dentatus and Lavandula officinalis to harsh winters across North Western Himalaya Sumira Jan, Azra N Kamili, Javid A Parray and Yashbir Singh Bedi Centre of research for Development, University of Kashmir, Srinagar-190006 Plant Biotechnology Division, CSIR - Indian Institute of Integrative Medicine, Canal Road, Jammu – 180 001
ABSTRACT Herbs adapt to diverse climates exhibit distinct variability to fluctuating temperatures and demonstrated various metabolic and physiological adaptations to harsh environment. In present research Rumex dentatus and Lavandula officinalis were collected before snowfall in SeptemberNovember to evaluate variability in major phytoconstituents to diverse seasonal regime. The LCMS was used for simultaneous determination of eight anthraquinone derivatives in R. dentatus i.e., emodin, physcion, chrysophanol, physcion glucoside, endocrocin, emodin glucoside, chrysophanol glucoside, chromone derivatives and in L. officinalis monoterpenes terpenes i.e., (Z)-b-Ocimene, E)-b-Ocimene, terpene alcohol, Terpin-4-ol, acetate ester-Linalyl acetate and bicyclic sesquiterpeniod (E)-caryophyllene. The correlation analysis confirmed significant variation in anthraquinone glycoside and terpene content within Rumex and lavender respectively and altitude was established as determinant factor in secondary metabolism of both herbs. The study concludes the propagation of herbs in bioclimatic belts which favor accumulation of major constituents and validates their greater pharmacological activity. Key words: Rumex dentatus, LC-MS, Anthraquinone glucoside, Monoterpene, Quantitative analysis, Environmental factor
Experimental Survey and Collection of plant material at various altitudes Three sites from sub-alpine areas with dense herbal diversity from varied altitudes of Western Himalayas with different snow fall coverage were selected as characterized in Table S1. These collection sites are previously documented by Kaul (1997) for their abundant herbal wealth. Plant Extraction The different vegetative and reproductive parts of two herbs were procured at respective stages and then air-dried (for 30 days). The fully expanded leaves (1.5g) from each herb were then powdered and sieve through mesh to get a fine powder (100-150 mesh). The powder 0.25g was weighed, distilled for 1.5 h in 100 mL of water in a soxhlet apparatus. The plant material was pre-extracted with petroleum ether to remove oily substances. Sample preparation for free anthraquinone and anthraquinone glucosides isolation: The resulting petroleum ether extract was then refluxed with ethyl acetate and chloroform followed by 10 ml of methanol in ultra sonic water bath at 25 °C for 30 min. The solution was filtered through 0.2 µm membranes before use, and 3µl aliquot was injected into LC for analysis. Sample preparation for monoterpene isolation: For the monoterpene isolation method previously described by Boira & Blanquer 1998; MunozBertomeu et al. 2006 was modified to validate its accuracy for LC-MS method. The resulting petroleum ether extract was refluxed with hexane containing n-tetradecane and naphthalene as internal standards and dried over anhydrous sodium sulphate. The resulting solution was then filtered through 0.22 mm polyvinylidene difluoride Millipore membranes, and adjusted to a final volume of 10 or 50 ml with hexane to obtain 10 mg/ml n-tetradecane and 400 mg/mL naphthalene or 2 mg/ml n-tetradecane and 80 mg/ml naphthalene for leaves or flower distillates, respectively. The final aliquot were kept in airtight glass containers stored at 4 °C until further use. For quantification of the metabolites, the products were quantified (mg/g dried tissue) by comparison of detector response with that of the internal standards, assuming equal response factors. Also, percentages of compounds were determined from their peak areas. The relative peak area for individual constituents was determined using the Chrom-Card S/W program (Thermo, Finnigan). All analyses were performed at least four times.
Instrumentation The LC system (Waters, Milford, MA) consisted of a pump equipped with a 600E system controller, autosampler 717, and dual UV detector 2487. Data were processed with Empower 2 software.
Water containing mobile phases was filtered through a 0.22µM GS filter (Millipore, UK) and degassed in an ultrasonic bath for 10 min before use. The gradient mobile phases were degassed continuously by sparking with helium at a rate of 40 ml min−1. UV detection was carried out at 302 and 464 nm, respectively. Gemini 5µ C18 column (250mm×4.6mm, Phenomenex) with a guard column was used for the isolation of individual AQs and alkaloids. Mobile phase consisted of water (A) and methanol (B), both containing 1% TFA. Gradient elution started at 30% B (0 min), increasing linearly to 100% B within 40 min. Each analysis was followed by a column washing (100% B, 10 min) and equilibration step (15 min), resulting in total analysis time 65min. The flow rate was kept at 1.0 ml min−1. Fractions containing individual compounds were collected, evaporated to dryness under reduced pressure and used for further FTMS analysis. The Kinetex 2.6µ C18 column (150mm×4.6mm, Phenomenex) was used for the HPLC method development. Gradient elution (0 min, 20% B; 30 min, 50% B) with mobile phase consisting of phosphate buffer (50mM; pH= 2.0)/ acetonitrile (9:1, v/v; solvent A) and acetonitrile (solvent B); flow rate, 0.7 ml min−1; injection volume, 3µl; UV detection at 302 nm. Fractions containing individual compounds were desalted and used for detailed FTMS analysis. Mass spectrometry Mass spectrometric (MS) experiments were performed on a Fourier transform ion cyclotron resonance instrument (FTMS) (9.4T APEX-Ultra, Bruker Daltonics, Billerica, MA). The instrument was operated in a negative ion mode. Spectra were collected over the mass range 150–2000 m/z at 1M data points resulting in a maximum resolution of 200,000 at 400 m/z. Dried samples were dissolved in 1ml of MeOH–H2O (1:1,v/v), diluted 50× and introduced to MS by direct infusion via electro spray ion source. The flow rate was 1.5µlmin−1 and the temperature of drying gas (nitrogen) was set to 230 0C. The species of interest were isolated in the gas phase with a 3.0 m/z window and fragmentation was induced by dropping the potential of the collision cell (16–22V depending on the compound). The accumulation time was set at 0.5 s, the cell was opened for 1200s, 8 experiments were collected for each spectrum. The instrument was externally calibrated using singly charged arginine clusters resulting in sub ppm accuracy.
PCA analysis The constituents of Rumex dentatus and Lavandula officinalis were evaluated across the sites as well as in the sites by principal component analysis (PCA) using SPSS 17.0 software.
Table S1. Sites for collection of wild herbs in Western Himalayan Ranges Character
Site I Pisu top (Pahalgam)
Site II Mount Apharwat (Gulmarg)
Altitude Forest Range Climatic Zones Direction Latitude Longitude Snow fall (2012)
4,175m Upper Dachigam Sub-alpine South East 34° 0’ 36’N, 75° 11’ 24’ E 244cm Dense
4,100 m Pir Panjal Temperate-subalpine North West 34°03′N 74°23′E 34.05°N 74.38°E 308cm Dense
Figure S1: PCA analysis of different constituents and their derivatives of R. dentatus and L. polyphllus at different sites. S1a- PCA analysis among anthraquinone derivatives of R. dentatus across sites S1b- Relationship between sites in terms of performance for constituents of R. dentatus S1c- Relationship of different constituents in different sites of R. dentatus S1d- PCA analysis among monoterpenes of L. officinalis across sites Interpretation: Lesser the angle- Strong positive correlation. Greater the angle-strong negative correlation. Length of the line which designates a particular constituent determines its variation across sites. More length greater variation & Small length less variation across sites
Figure S2: LC- UV chromatograms of (a) Rumex dentatus and, (b) Lavandula officinalis
Figure S3: ESI-MS/MS of free anthraquinones and anthraquinone glucosides in Rumex dentatus
Figure S4: ESI-MS/MS of monoterpenes in L. officinalis
References: Boira, H., & Blanquer A. (1998). Environmental factors affecting chemical variability of essential oils in Thymus piperella L. Biochemical Systematics and Ecology, 26, 811-822. Kaul, M.K. (1997). Medicinal plants of Kashmir and Ladakh, temperate and cold-arid Himalaya. New Delhi: Indus Publishing Co; p. 173. Munoz-Bertomeu, J., Arrillaga, I., Ros, R., & Segura J. (2007). Essential oil variation within and among natural populations of Lavandula latifolia and its relation to their ecological areas. Biochemical Systematics and Ecology, 35, 479-488.
ABSTRACT Herbs adapt to diverse climates exhibit distinct variability to fluctuating temperatures and demonstrated various metabolic and physiological adaptations to harsh environment. In present research Rumex dentatus and Lavandula officinalis were collected before snowfall in SeptemberNovember to evaluate variability in major phytoconstituents to diverse seasonal regime. The LCMS was used for simultaneous determination of eight anthraquinone derivatives in R. dentatus i.e., emodin, physcion, chrysophanol, physcion glucoside, endocrocin, emodin glucoside, chrysophanol glucoside, chromone derivatives and in L. officinalis monoterpenes terpenes i.e., (Z)-b-Ocimene, E)-b-Ocimene, terpene alcohol, Terpin-4-ol, acetate ester-Linalyl acetate and bicyclic sesquiterpeniod (E)-caryophyllene. The correlation analysis confirmed significant variation in anthraquinone glycoside and terpene content within Rumex and lavender respectively and altitude was established as determinant factor in secondary metabolism of both herbs. The study concludes the propagation of herbs in bioclimatic belts which favor accumulation of major constituents and validates their greater pharmacological activity. Key words: Rumex dentatus, LC-MS, Anthraquinone glucoside, Monoterpene, Quantitative analysis, Environmental factor
Experimental Survey and Collection of plant material at various altitudes Three sites from sub-alpine areas with dense herbal diversity from varied altitudes of Western Himalayas with different snow fall coverage were selected as characterized in Table S1. These collection sites are previously documented by Kaul (1997) for their abundant herbal wealth. Plant Extraction The different vegetative and reproductive parts of two herbs were procured at respective stages and then air-dried (for 30 days). The fully expanded leaves (1.5g) from each herb were then powdered and sieve through mesh to get a fine powder (100-150 mesh). The powder 0.25g was weighed, distilled for 1.5 h in 100 mL of water in a soxhlet apparatus. The plant material was pre-extracted with petroleum ether to remove oily substances. Sample preparation for free anthraquinone and anthraquinone glucosides isolation: The resulting petroleum ether extract was then refluxed with ethyl acetate and chloroform followed by 10 ml of methanol in ultra sonic water bath at 25 °C for 30 min. The solution was filtered through 0.2 µm membranes before use, and 3µl aliquot was injected into LC for analysis. Sample preparation for monoterpene isolation: For the monoterpene isolation method previously described by Boira & Blanquer 1998; MunozBertomeu et al. 2006 was modified to validate its accuracy for LC-MS method. The resulting petroleum ether extract was refluxed with hexane containing n-tetradecane and naphthalene as internal standards and dried over anhydrous sodium sulphate. The resulting solution was then filtered through 0.22 mm polyvinylidene difluoride Millipore membranes, and adjusted to a final volume of 10 or 50 ml with hexane to obtain 10 mg/ml n-tetradecane and 400 mg/mL naphthalene or 2 mg/ml n-tetradecane and 80 mg/ml naphthalene for leaves or flower distillates, respectively. The final aliquot were kept in airtight glass containers stored at 4 °C until further use. For quantification of the metabolites, the products were quantified (mg/g dried tissue) by comparison of detector response with that of the internal standards, assuming equal response factors. Also, percentages of compounds were determined from their peak areas. The relative peak area for individual constituents was determined using the Chrom-Card S/W program (Thermo, Finnigan). All analyses were performed at least four times.
Instrumentation The LC system (Waters, Milford, MA) consisted of a pump equipped with a 600E system controller, autosampler 717, and dual UV detector 2487. Data were processed with Empower 2 software.
Water containing mobile phases was filtered through a 0.22µM GS filter (Millipore, UK) and degassed in an ultrasonic bath for 10 min before use. The gradient mobile phases were degassed continuously by sparking with helium at a rate of 40 ml min−1. UV detection was carried out at 302 and 464 nm, respectively. Gemini 5µ C18 column (250mm×4.6mm, Phenomenex) with a guard column was used for the isolation of individual AQs and alkaloids. Mobile phase consisted of water (A) and methanol (B), both containing 1% TFA. Gradient elution started at 30% B (0 min), increasing linearly to 100% B within 40 min. Each analysis was followed by a column washing (100% B, 10 min) and equilibration step (15 min), resulting in total analysis time 65min. The flow rate was kept at 1.0 ml min−1. Fractions containing individual compounds were collected, evaporated to dryness under reduced pressure and used for further FTMS analysis. The Kinetex 2.6µ C18 column (150mm×4.6mm, Phenomenex) was used for the HPLC method development. Gradient elution (0 min, 20% B; 30 min, 50% B) with mobile phase consisting of phosphate buffer (50mM; pH= 2.0)/ acetonitrile (9:1, v/v; solvent A) and acetonitrile (solvent B); flow rate, 0.7 ml min−1; injection volume, 3µl; UV detection at 302 nm. Fractions containing individual compounds were desalted and used for detailed FTMS analysis. Mass spectrometry Mass spectrometric (MS) experiments were performed on a Fourier transform ion cyclotron resonance instrument (FTMS) (9.4T APEX-Ultra, Bruker Daltonics, Billerica, MA). The instrument was operated in a negative ion mode. Spectra were collected over the mass range 150–2000 m/z at 1M data points resulting in a maximum resolution of 200,000 at 400 m/z. Dried samples were dissolved in 1ml of MeOH–H2O (1:1,v/v), diluted 50× and introduced to MS by direct infusion via electro spray ion source. The flow rate was 1.5µlmin−1 and the temperature of drying gas (nitrogen) was set to 230 0C. The species of interest were isolated in the gas phase with a 3.0 m/z window and fragmentation was induced by dropping the potential of the collision cell (16–22V depending on the compound). The accumulation time was set at 0.5 s, the cell was opened for 1200s, 8 experiments were collected for each spectrum. The instrument was externally calibrated using singly charged arginine clusters resulting in sub ppm accuracy.
PCA analysis The constituents of Rumex dentatus and Lavandula officinalis were evaluated across the sites as well as in the sites by principal component analysis (PCA) using SPSS 17.0 software.
Table S1. Sites for collection of wild herbs in Western Himalayan Ranges Character
Site I Pisu top (Pahalgam)
Site II Mount Apharwat (Gulmarg)
Altitude Forest Range Climatic Zones Direction Latitude Longitude Snow fall (2012)
4,175m Upper Dachigam Sub-alpine South East 34° 0’ 36’N, 75° 11’ 24’ E 244cm Dense
4,100 m Pir Panjal Temperate-subalpine North West 34°03′N 74°23′E 34.05°N 74.38°E 308cm Dense
Figure S1: PCA analysis of different constituents and their derivatives of R. dentatus and L. polyphllus at different sites. S1a- PCA analysis among anthraquinone derivatives of R. dentatus across sites S1b- Relationship between sites in terms of performance for constituents of R. dentatus S1c- Relationship of different constituents in different sites of R. dentatus S1d- PCA analysis among monoterpenes of L. officinalis across sites Interpretation: Lesser the angle- Strong positive correlation. Greater the angle-strong negative correlation. Length of the line which designates a particular constituent determines its variation across sites. More length greater variation & Small length less variation across sites
Figure S2: LC- UV chromatograms of (a) Rumex dentatus and, (b) Lavandula officinalis
Figure S3: ESI-MS/MS of free anthraquinones and anthraquinone glucosides in Rumex dentatus
Figure S4: ESI-MS/MS of monoterpenes in L. officinalis
References: Boira, H., & Blanquer A. (1998). Environmental factors affecting chemical variability of essential oils in Thymus piperella L. Biochemical Systematics and Ecology, 26, 811-822. Kaul, M.K. (1997). Medicinal plants of Kashmir and Ladakh, temperate and cold-arid Himalaya. New Delhi: Indus Publishing Co; p. 173. Munoz-Bertomeu, J., Arrillaga, I., Ros, R., & Segura J. (2007). Essential oil variation within and among natural populations of Lavandula latifolia and its relation to their ecological areas. Biochemical Systematics and Ecology, 35, 479-488.
