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THE ORIGINS


Design & Content: PoliNat | Polifenoles Naturales

First edition: March 2013

Total or partial reproduction of this work by any means or process, including (but not restricted to) photocopying and computer processing, and distribution of copies of it by rental or public lending, and the export or import of such copies for distribution for sale is strictly prohibited without written permission from the copyright holders, under penalties provided by the law. © PoliNat | Polifenoles Naturales S.L. C/ Taibique 4 · Polígono Industrial Las Majoreras Las Palmas GC · España www.PoliNat.com


INDEX RAW MATERIALS ABOUT THE ORIGIN BOTANICAL IDENTITY THE REGION

THE PROCESS COLLECTION

GOOD COLLECTION PRACTICES PERMISSION TO COLLECT COLLECTION MANAGEMENT PLAN COLLECTION PRACTICES TIME OF COLLECTION

PERSONNEL PROCESSING

PRIMARY PROCESSING SECUNDARY PROCESSING

ENVIRONMENTAL PRACTICES & SUSTAINABILITY PROGRAMMES

CONCLUSION

PROCESSING GMP EFFICIENT PROCESSING FLOW CHART HIGH QUALITY

RHODIOLIFE PHYTOCHEMICAL PHENYLALKANOIDS PHENYLETHANOIDS PHENYLPROPANOIDS

CHROMATOGRAPHY EFFICACY & SAFETY

REFERENCES


RhodioLife

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About the Origin

ABOUT THE ORIGIN RHODIOLIFE IS DIFFERENT FROM THE VERY BEGINNING. STARTING WITH THE SIBERIAN ORIGIN, WILDCRAFTING, ROOTS SELECTION, PROGRAMMES FOR SUSTAINABILITY AND SOCIAL RESPONSIBILITY.

BOTANICAL IDENTITY Botanical identity is essential. Because of significant species-dependent variation in phytochemistry and pharmacology, the use of “Rhodiola” as a general term is inaccurate and misleading. The correct identification of all Rhodiola species according to precise and generally accepted botanical, phytochemical, and genetic taxonomic criteria is not merely an abstract intellectual exercise. It is critical for both scientific and phytopharmacological accuracy, as well as for product labelling for the public. The pharmacological and medicinal properties of Rhodiola are species dependent phenomena. Of all the Rhodiola species, Rhodiola rosea has been the predominant subject of phytochemical, animal, and human studies.

CLASS

Magnoliopsida

SUPERORDER

Saxifraganae

ORDER

Saxifragales

FAMILY

Crassulaceae – stonecrops, orpins

GENUS

Rhodiola L. – stonecrop

SPECIES

Rhodiola rosea L. – roseroot stonecrop

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RhodioLife

THE REGION While Rhodiola as a genus may have originated in the mountainous regions of Southwest China and the Himalayas, botanists have established that Rhodiola rosea naturally display a circumpolar distribution in mountainous regions in the higher latitudes and elevations of the Northern Hemisphere. In Central and Northern Asia, the genus is distributed from the Altai Mountains across Mongolia into many parts of Siberia. Rhodiola rosea used for the production of RhodioLife is wildcraft collected, under the Russian Government License, from the regions of Ust’-Kan, Charysh, Ust’Koksa, Altay, 450 km southwest of Novosibirsk, in the Altai State. Altay Mountains represents a pristine area free from contamination in of the most well preserved and remote natural environments.

Russia Mongolia Kazakhstan

China

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About the Origin

Altái Republic [Russia]

Ust Koksa Charysh

Ust-Kan

Key facts about Altái One of the meanings of the word Altai is “Golden Mountains” (from the Mongolian Word “altan”) Forests cover more than half of the Mountain territory. There are more than 2000 species of supreme vascular plants and approximately 200 kinds of vegetable plants A calculated total area of 728 km2 of lakes is distributed amongst more than 7000 lakes in the region. The calculated amount of glacial water in the region is approximately 52km3 and there are more than 60000 km worth of waterways. Altai has a temperate continental climate with short hot summers and long cold, frosty winters. Average yearly temperature is approximately 0ºC with 50-700 mm annual rainfall. The area is regulated by elaborated and established norms of recreational exploitation to ensure preservation of rare plants. In fact more than 60% of the surface is composed of strictly protected areas.

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RhodioLife

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About the Origin

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RhodioLife

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The Process

THE PROCESS 1- COLLECTION

PoliNat closely collaborates with our supplier implementing operating procedures that provide general technical guidance for the sustainable collection and processing of Rhodiola rosea roots, following the overall context of quality assurance and the WHO guidelines on Good Agricultural and Collection Practices (GACP). GACP provides a basis for a quality assurance system for the starting Rhdodiola rosea roots used in the production of RhodioLife. This includes the collection of the plants in the wild, as well as primary processing of the plant material, such as drying, packaging in bulk, storage and transport of the raw materials until it arrives to the PoliNat factory. The impact of collection on the environment and ecological processes, and the welfare of local communities are also considered. GACP apply to the first part of the production, for which Good Manufacturing Practice (GMP) does not apply yet. Applying for GACP eliminates or reduces the risks of microbiological or chemical contamination, mistaken identity, and deterioration during primary processing and storage. Eliminating and reducing these risks enhance the reliability of the starting Rhodiola rosea roots used in the production of RhodioLife. PoliNat works with a local company based in Byisk (our supplier), which is responsible for the Collection Management Plan. This Plan integrates all the aspects related not only to the GACP but also to the regulatory and permission necessary for the legal export of Rhodiola rosea raw materials from Russia.

GOOD COLLECTION PRACTICES Application of GACP is primarily intended to provide a general technical guidance on obtaining Rhodiola rosea roots of high quality for the sustainable production of RhodioLife. Applying for Good Collection Practices pretends to ensure the long-term survival of Rhodiola rosea wild populations and their associated habitats.

PERMISSION TO COLLECT Permission for collecting plants is given by the management of the Wood Industry of Altay Region after demand coordination in the Ministry of Natural Resources of the Russian Federation, which is also responsible for issuing the Export Licenses. Collecting permissions are strictly connected to export licences to prevent over collecting of Rhodiola roots. Polifenoles Naturales, SL (PoliNat) is one of the few companies granted with an Export License by the Russian Government. Collection areas and crafting activities are supervised by the Federal Office for Natural Resource Exploitation (FONRE). PoliNat audits regularly (every two years) our supplier facilities and the whole process. The last audit was conducted in July, 2012. In this audit, an independent auditor from ECOCERT audited the wild crafting activities.

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RhodioLife

COLLECTION MANAGEMENT PLAN Collection activities are supervised by the FOREN. The collection management Plan includes the following requisites:

�Government

Collection permission from Russian Government

�Government supervision of Collection Activities

�Geographical location of the collection sites.

Location & collection

Approximate plant density and maximum allowed collection volumes.

- Ministry of Natural Resources of the Russian Federation

- Federal Office for Natural Resource Exploitation. Altay Republic. - Export License: Issued to Polifenoles Naturales, SL by the Russian Government.

- Specifically designated in the Areas of Ust’-Kan, Charysh, Ust’-Koksa, Altay, 450 km southwest of Novosibirsk (Altay Republic) - Altitude: 1500-2000m above sea level.

- Determined by the FOREN. Never exceeds 50% of the exiting natural density

Essential information about Rhodiola for plant identification

Worker training in collection activities and plant identification

Environmental information

- Taxonomy - Distribution - Phenology

- Topography - Soil - Climate

- Genetic diversity - Reproductive biology

- Vegetation - Prospective collecting site(s)

Procedures

Processing procedures

Transport

Collection Practices

Transport planning for personnel, equipment, supplies and collected plant material

THE COLLECTION PROGRAMME ENSURES THERE IS NO IMPACT ON:

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- Local communities - Ecological impact - The stability of the natural habitat - The maintenance of sustainable populations of Rhodiola plants in the collection area(s)


The Process

PERSONNEL Collectors are organized in brigades (3 – 10 workers) and are transported to the collection fields up the mountains, where they live from mid-May till the end of September (about 4 months). They are familiar with good collecting techniques, transport, and handling of equipment and plant materials, including cleaning, drying and storage. Training of personnel is conducted regularly. There are local experts (brigade leaders) responsible for the field collection with practical education and training in Rhodiola rosea biology and plant identification, with practical experience in fieldwork. They are also responsible for the supervision of workers and the full documentation of the work performed. Field personnel should have adequate botanical training, and be able to recognize medicinal plants by their common names and, ideally, by their scientific (Latin) names. Collectors should also receive instructions on all issues relevant to the protection of the environment and the conservation of plant species, as well as the social benefits of sustainable collection of medicinal plants. Collectors are instructed for addressing three personnel important issues: training, safety and hygiene. This is relevant to all phases of collecting and post-harvest handling.

TRAINING In collection and post-harvest activities. In the positive identification of Rhodiola rosea plants. In proper hygienic practices with specific attention to preventing microbial contamination of handled Rhodiola.

SAFETY Clothing. Ensure that personnel wear clothing and shoes that provide protection that is appropriate to the work environment. Environmental factors. Consider and establish procedures to protect personnel from environmental factors that are relevant to worker safety. Tools and equipment. Maintain all tools, equipment and vehicles used by personnel to ensure that these will be reasonably expected to be reliable and safe.

HYGIENE Prevention of contamination. Provide toilets, hot running water and soap at postharvest handling facilities. Establish minimum hand washing requirements. Sick personnel or those with open wounds/skin infections are not allowed to work. RhodioLife ¡ Product Developed & Manufactured by PoliNat | 13


RhodioLife

COLLECTION PRACTICES All the collection practices are designed to guarantee the long-term survival of wild populations and their associated habitats. There are Standard Operating Procedures (SOPs) which clearly describe the work to be done during the collection of the plants, the processing and the transport. A traceability system allows the tracking of all raw materials received in the final processing and storage facilities.

TIME OF COLLECTION It is well known that the quantitative concentration of biologically active constituents varies with the stage of plant growth and development. This also applies to Rhodiola. The best time for collection of Rhodiola rosea has been determined according to the quality and quantity of biologically active constituents. Ecologically non-destructive systems of collection are employed. Only plants over 5 years old are collected. This can be easily determined by the root size verification (root weight over 300 grs) which involves the removal of the superficial soil that covers the aerial part. This ensures the best possible quality in terms of the balance and content of bioactive markers. Only 50% of the plant population in a crafting area is collected. Collected roots are placed in clean baskets, mesh bags or other well aerated containers that are free from foreign matter.

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The Process

ROOT WEIGHT (gr)

1 year / 50 gr 2 years / 124 gr 3 years / 191 gr 5 years / 312 gr 5 years

10 years / 560 gr

312 gr

BIOACTIVES %

1 year / 1,19 % 2 years / 1,26 % 3 years / 1,31 %

5 years 1,52 %

5 years / 1,52 % 10 years / 1,32

ACTIVES RATIO / YEAR 1

1 year / 0,60

0,75

Ratio

2 years / 0,78 0,50

3 years / 0,8

5 years / 0,95 0,25

10 years / 0,74

0 1 year

2 years

3 years

5 years

10 years RhodioLife 路 Product Developed & Manufactured by PoliNat | 15


RhodioLife

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The Process

THE PROCESS 2- PROCESSING PROCEDURES Rhodiola rosea extract processing follows the indications given by the Russian Pharmacopoeia. After collection, the Rhodiola rosea is subjected to appropriate preliminary processing, including elimination of undesirable materials, the excess of soil, sorting and cutting. The collected Rhodiola rosea roots are protected from insects, rodents, birds and other pests, and from livestock and domestic animals.

Collecting tools, such as machetes, shears, saws and mechanical tools, are kept clean and maintained in proper condition. Those parts that come into direct contact with the collected medicinal plant materials should be free from excess oil and other contamination. As the collection sites are located at large distances from the consolidation/processing facilities, the process is divided in two main steps.

PRIMARY PROCESSING (AT THE COLLECTION SITES)

COLLECTION

CLEANING

CUTTING

DRYING

PACKAGING

TRANSPORT

SECONDARY PROCESSING (AT THE CONSOLIDATION SITE)

MANUAL CLEANING

SIEVING

DRYING

FINAL PACKAGING

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RhodioLife

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The Process

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RhodioLife

ENVIRONMENTAL PRACTICES AND SUSTAINABILITY PROGRAMMES

Collection practices applied by the collectors to the harvest of Rhodiola rosea address not only their need to gain economic benefits from the sale of the harvested plants, but also to make sure that Rhodiola rosea survives. In addition to preserving plant populations, harvest practices are oriented to minimize the damage to the local habitat. Re-harvesting in the same location is restricted to every 5 years to allow sufficient reestablisment of the plant population. Furthermore, only 50% of the population from a specific location can be collected so that enough plants able to produce an adequate amount of seeds to sustain the population are kept. Since Rhodiola can regenerate through vegetative growth, a portion of the root is left in the ground for plant regeneration. A program for the cultivation and replantation of Rhodiola plants in the harvested locations has been implemented over the past three years. At the present moment, this project grows up to 5.000 Rhodiola plants/year.

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CONCLUSION The use of basic standardized procedures contributes to the batch-to-batch conformity and thus, to the reliability and high quality of Rhodiola rosea starting raw material. The application of GACP significantly contributes to improve the quality of starting raw materials before PoliNat’s GMP system takes the lead in the production of RhodioLife.

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RhodioLife

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Processing

PROCESSING RHODIOLIFE IS THE RESULT OF APPLYING CAREFUL MANUFACTURING PROCEDURES TO A HIGH QUALITY MATERIAL. SKILLFULL AND THOROUGH IMPLEMENTATION OF GMP AND STRICT QUALITY CONTROL PROGRAMS ENSURES DELIVERY OF A UNIQUE PRODUCT.

GMP PoliNat’s GMP management system (Poli 360) takes the lead. It starts with a careful inspection of raw materials at the site of production ensuring that sustainable, eco-friendly and social responsible practices are at the foundation of our suppliers. Our GMP system starts before we receive the raw materials with the audit and approval of our suppliers. More than 15 years of continued, committed work and accumulated experience working with our Siberian Rhodiola rosea root supplier is the base for the safety and consistency in the quality of the starting raw material for the production of RhodioLife.

aspects related to a product quality and safety but to all activities within the company. Every process that has an impact on product quality is precisely documented and available for review. Only Purified Water and/or Water : Food Grade Ethanol are used as solvents for the production of RhodioLife. Extraction, separation and concentration applied technologies, carefully preserve the natural phytochemical profile and main bioactive compounds responsible for the health benefits of this nature’s gift.

EFFICIENT PROCESSING Our highly experienced staff takes good care of all approved materials, closely watching an industrial production process that ensures a high concentration of bioactives according to our clients’ needs and expectations. High quality of the entire in-house process is maintained by a constant interaction between the QA team and the physico-chemical laboratory, a state of the art equipped facility where a professional team can perform exact quantifications and characterizations of bioactives. All manufacturing steps follow strict Good Manufacturing Practices. Well defined procedures and analysis at critical steps of production guarantee the quality and consistency in all manufacturing process. All raw data and critical parameters are recorded in a Master Batch Record (MBR) which allows the revision of all the processes and product traceability along all manufacturing steps. PoliNat facilities and production process strictly comply with GMPs requisites. We are committed to continuous improvement of Quality Assurance (QA) which is actively engaged in improving not only those

PRESERVATION OF THE NATURAL ACTIVE INGREDIENTS THROUGH SMART, EFFICIENT PROCESSING. RhodioLife · Product Developed & Manufactured by PoliNat | 23


RhodioLife

FLOW CHART Rhodiola Root

Quality Control

Pressure, Temperature, Time

Process

Extraction

Filtration

Appearance, Homogeneity, Temperature, Time, Loss of drying, Content of bioactives

Distillation at low Temperature under reduced Pressure

Concentration

Appearance, Homogeneity, Temperature, Time, Loss of drying, Content of bioactives

IPC: Appearance & Granulometry

IPC: Appearance, Net & Gross weights

QC: See CoA

Drying

Sieving (80 Mesh)

Weighing and packaging

Analysis

* IPC: Internal Process Control * QC: Quality Control

TM

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Processing

HIGH QUALITY All RhodioLife lots are precisely inspected before shipping and require production clearance by QA before being released. At reception, a close contact with customers gives us the final assurance that a high quality product has been successfully delivered. Additional quality control checks are implemented under specific requirements to meet the national regulatory demands as requested by customers to ensure they receive a full compliant product approved for human consumption in the country where the ingredient will be sold as final product. The most advanced technology and analytical instrumentation, including UPLC-DAD/MS or GC-MS according to suitable validated methods are employed in the analysis of our products. Only qualified analytical reference standards with certified origin are used for this purpose. All data are summarized in the Certificate of Analysis which must strictly comply with product specifications.

ANALYTICAL CONTROL IN FINISHED PRODUCTS 1. Concentration of bioactives 2. Moisture 3. Heavy metals (Totals, Pb, As, Hg, Cd) 4. Mycotoxyns (Total Aflatoxin) 5. Microbiology (Aerobics, Yeast & Mold, E. Coli, Enterobacteriae, S. aureus, Listeria and Salmonella) 6.Pesticides

EXTERNAL CERTIFICATIONS Company

Products *

*

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RhodioLife

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Phytochemical

PHYTOCHEMICAL COMPOSITION Adaptogenic activity related to Rhodiola rosea has been traditionally attributed to the presence of four principal active ingredients: salidroside, rosin, rosavin and rosarin (Sokolov, 1985; Furmanowa, 1998). These compounds are found at relatively high concentrations in the roots of this plant (> 0.5% - 3 %; w: w), and are frequently employed for the standardization of commercial products (Markus, 2001; Panossian, 2005; Yousef, 2006). Extracts used in most clinical trials are standardized to minimum 3% total rosavins and 0.8-1% salidroside, as the naturally occurring ratio of these compounds in the plant rhizomes is approximately 3:1 (Brown, 2002).

Phenolic Acids

Other biologically active substances found in Rhodiola rosea include flavonoids (rhodiolin, rhodionin, rhodiosin, tricin, rhodalgin and acetylrhodalgin), monoterpenes (rosiridol and rosiridin), phenylmethanoids (benzyl alcohol O-αL-arabinopyranosyl- (1→6)-β-D-glucopyranosyde and phenyl methyl O-α-L-arabinofuranosyl-(1→6)β-D-glucopyranosyde), phenolic acids (gallic acid, chlorogenic acid, caffeic acid and hydroxycinnamic acid) and cyanogenic glycosides (rhodiocyanoside A and lotaustralin) (Ma, 2006; Avula, 2009).

BIOACTIVES PHENYLALKANOIDS

- PHENYLETHANOIDS - PHENYLPROPANOIDS

Cyanogenic Glycosides

Terpenoids

Rhodiola rosea Flavonoids

Phenylalkanoids

Phenylpropanoids Rosin Rosavin Rosarin

Phenylmethanoids

Phenylethanoids Salidroside

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RhodioLife

PHENYLALKANOIDS PHENYLETHANOIDS Principal phenylethanoids found in Rhodiola species consist of glycosides produced by biotransformation of the phenylpropanoid tyrosol (Fig. 1). Among all of these compounds, salidroside has been reported as the most active tyrosol glycoside, being associated, together with rosavins, to the anti-depressive and anxiolytic effects observed in golden root (Maslowa, 1994; Tolonen, 2003).

Other phenylethanoids have also been isolated from R. rosea, such as mongrhoside, viridioside, or 2-phenylethyl vicianoside. However, the scientific literature concerning the bioactivity of these substances is limited (e.g. Jim茅nez 1994).

> Salidroside Tyrosine tyrosine decarboxylase Fig 2. // Chemical structure of salidroside.

Tyramine tyramine oxydase

4-hydroxy-phenylacetaldehyde

aryl alcohol dehydrogenase

Tyrosol

+ glucose

Salidroside Fig 1. // Biosynthetic pathway of salidroside.

Some authors have stated that salidroside could be biologically more active than rosavin (Panossian, 2008). However, it is suspected that the beneficial effects reported for R. rosea are probably due to the presence of rosavins or to the synergetic effect of rosavins and salidroside (Tolonen, 2004; Perfumi, 2007).

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Phytochemical

As mentioned before, the main compounds responsible for R. rosea activity are believed to belong to this family, which include phenylethanoids and phenylpropanoids.

PHENYLPROPANOIDS Rosavins are products of the phenylpropanoid metabolism, derived from phenylalanine, which is produced in the shikimicchorismic acid pathway (Fig. 3). Rosin is formed from the condensation of one glucose unit with cinnamyl alcohol and represents the simplest phenylpropanoid glycoside found in Rhodiola rosea. The diglucosides rosavin and rosarin are metabolized from rosin and are formed by adding an extra glycoside unit of arabinose and arabinofuranose respectively. Depending on the sugar type and the site it is connected to, further glycosides may be formed (György, 2004; György, 2005).

While phenylethanoid derivatives, such as salidroside, are present in all species of the Rhodiola genus (and even outside this genus), phenylpropanoid derivatives, such as the rosavins (rosavin, rosin, rosarin), are specific components of R. rosea (Kurkin, 1986; Ramazanov, 1999). The chemical structures of these compounds are shown below (Fig. 4).

> Rosarin

Phenylalanine

tyrosine decarboxylase

> Rosavin Trans-cinnamate

tyramine oxydase

> Rosin Cinnamyl-CoA

aryl alcohol dehydrogenase

Cinnamaldehyde

Fig 4. // Chemical structures of the rosavins: rosarin, rosavin and rosin.

+ glucose

Cinnamyl alcohol

+ arabinose

Rosin

Rosavin

+ arabinofuranose

Rosarin

Fig 3. // Biosynthetic pathway of rosavins.

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RhodioLife

CHROMATOGRAPHY PoliNat pays rigorous attention to the identification and precise quantification of the levels of the main active ingredients (salidroside, rosin, rosavin and rosarin) found in their Rhodiola rosea related products. RhodioLife is the standardized Rhodiola rosea extract manufactured by PoliNat, standardized to a minimum of 3 or 5% total rosavins content. In line with the latest technologies, Ultrahigh Performance Liquid Chromatography (UHPLC) with Photodiode Array Detection (PDA) is used to provide high-resolution separation, identification and quantification of the main active ingredients found in Rhodiola rosea, ensuring product specifications are met. Figure 1. shows the chromatographic profiles obtained for the Rhodiola rosea roots employed as the raw material and a RhodioLife sample

standardized to 5% of total rosavins. An excellent correlation is observed between the Rhodiola Rosea root raw material and RhodioLife, indicating that the natural ratios between the different phytoactive compounds are preserved during the extraction and manufacturing process. Preservation of the natural balance between the different phytonutrients is essential to maintain the synergistic effect of the different phytoactive compounds. A recent study indicates large variations in the content of the marker compounds of the commercial Rhodiola Rosea powder extracts from 8 global suppliers, even among samples collected from the same supplier (Ma et al. 2011). This highlights the importance of careful standardization of the bioactive compounds in commercial Rhodiola rosea powder extracts.

Rosavin Rosin

AU

Salidroside Rosarin

Wavelength

3D chromatographic profile of Rhodiola rosea

Minutes

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Chromatography

RAW MATERIAL CHROMATOGRAPHY 0.25

Rosavin

0.20

0.15

AU

Rosarin Salidroside

0.10

Rosin

0.05

0.00 0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

3.50

4.00

4.50

5.00

Minutes

RHODIOLIFE CHROMATOGRAPHY 0.25

Rosavin 0.20

0.15

Rosarin

AU

Rosin

Salidroside 0.10

0.05

0.00 0.00

0.50

1.00

1.50

2.00

2.50

3.00

Minutes Fig 1. // Cromatographic profiles of Rhodiola rosea root raw material and RhodioLife 5% total rosavins.

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RhodioLife

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Efficacy & Safety

EFFICACY & SAFETY BACKGROUND Adaptation is required to successfully combat stressful situations, and has been described as “the ability to be exposed to a stressor while responding with either decreased or no characteristic neurohormonal perturbations”. Plant adaptogens cause our physiology to begin the adaptation process to stress and consuming adaptogens generates a degree of generalized non-specific resistance that allows our physiology to handle the stressful situation in a more resourceful manner (Brekham, 1969). Traditionally, the Siberians have used Rhodiola rosea to increase physical endurance, work productivity, longevity, and resistance to highaltitude sickness, and to treat fatigue and depression. Research, for the other hand, has revealed that it protected animals and humans against mental and physical stress, toxins, and cold (Kelly, 2001). Extracts of the roots have been found to contain powerful adaptogens (Ma, 2006; Kurkin, 1968; Furmanowa, 1998) Rhodiola rosea appears to offer an advantage over other adaptogens (i.e. Eleutherococcus senticosus or Panax ginseng) in acute phases of stress, in the sense that a single dose of Rhodiola rosea before acute stress seems to produce favorable results and prevents stress-induced disruptions. Because many stressful situations can be acute, unexpected in nature an adaptogen that can be taken acutely rather than chronically could be regarded as an advantage (Kelly, 2001; Khanum, 2005). There are many clinical studies and critical reviews conducted with various formulations of Rhodiola rosea extracts in different physical and psychological stress models, and the reader is refereed to those for more information (i.e.: Edokimov, 2009; Ishaque, 2012; Olsson, 2009; Parisi, 2010, Skarpanska-Steijnborn, 2009; Hung, 2011 to name a few).

RHODIOLIFE RESEARCH DATA Efficacy The effect of Rhodiola rosea (RR) supplementation on ATP content in muscles was studied in SpragueDawley rats. Briefly, 24 adult rats weighting 250 ± 20 g were divided 3 groups equally: control group, RR (50 mg / kg) and Rhodiola crenulata (50 mg / kg). Extracts and placebo were administered orally 30 minutes before performing forced swimming tests. Two sessions with 30-min intervals were carried out every day. The maximum duration of swimming was estimated on the basis of data recorded for 6 days. Results are summarized in figure 1: 7 6 5

*

4

*

**

*

*

**

3 2 1 0 Before test

After 6 days

After 24 h-rest

Control R. Crenulata R. rosea

Note: p<0.05 *compared with previous value **compared to control and R. crenulata groups

Fig 1. // ATP content in the mitochondria decreased after swimming

exercise in controls and animals of both experimental groups. However, the decrease in ATP content in rats receiving R. rosea extract was less pronounced in comparison (Abidov, 2003).

The authors concluded based on this study that Rhodiola rosea was” most effective for improving physical working capacity” (Abidov, 2003). RhodioLife · Product Developed & Manufactured by PoliNat | 33


RhodioLife

A subsequent clinical trial explored the possible benefits RR could bring to physical exercise in humans. A total of 36 healthy untrained volunteers (21-24 years of age) were randomly, equally divided in 3 groups (control, placebo and RR extract). Individuals in the placebo and RR extract groups received 2 capsules a day of each treatment (placebo or 340 mg of RR extract) for 30 days before and 6 days after exhausting physical exercise was. Results are summarized in figure 2: CRP 1,6 1,4 1,2 mg / l

*

*

1 0,8

*

0,6 0,4 0,2 0 Before test

5h post

5d post

Control Placebo R. rosea

Note: p<0.05 *compared with baseline levels **compared with levels 5h after test; +compared with control and placebo groups.

Ck 4000

** **

3500

U / ml

3000 2500

*

2000

*

*

*

1500 1000 500 0 Before test

5h post

5d post

Control Placebo R. rosea

Some authors have suggested that adaptation may be mediated by chaperon molecules such as heat shock proteins (HSP), which are fundamental in facilitating the cellular remodeling processes inherent to the training response. Published data indicate that acute endurance- and resistance-type exercise protocols increase the muscle content of several of these proteins, i.e.: HSP27, HSP60, HSC70 and HSP70 (Morton, 2009). We performed an in vitro experiment with myotubes to assess whether RhodioLife was able to elicit any response at the cellular level mediated by HSP70 (P茅rez, 2012). Myotubes were used as model of muscular tissue (Gorelick-Feldman, 2008) while oxygen peroxide was used as the stressor (increased oxidative stress).

*

*

The authors concluded that supplementation with the extract protected from physical exercise induced muscle damage and inflammation (Abidov, 2004).

Note: p<0.05 *compared with baseline levels **compared with levels 5h after test; +compared with control and placebo groups.

Fig 2. // Effect of supplementation wth Rhodiola rosea extract on

plasma CRP (C-Reactive Protein) and CK (Creatine Kinase) values in human volunteers (from Abidov, 2004).

34 | RhodioLife 路 Product Developed & Manufactured by PoliNat

In control cell cultures lacking pre-treatment, cell viability decreased 25 % following exposure to H2O2. In contrast, there was no significant decrease in cell viability when cells were pre-treated with RhodioLife at concentrations as low as 1 ppm for a period of up to 2 hrs. RhodioLife induced a 2-fold up-regulation and expression was further enhanced when pretreated cell cultures were exposed to H2O2 (6-fold), both with statistical significance (Fig. 3). HSP70 protein levels were maintained in cell cultures pretreated with RhodioLife compared to control cultures but expression was significantly lower (-50%) in cells lacking treatment and exposed to peroxide. Our results suggest that RhodioLife may enhance the production of HSP70 in muscles under conditions of increased oxidative stress such as intense physical exercise.


Efficacy & Safety

10

RHODIOLIFE EFFICACY SUMMARY:

*

9

Fold Regulation

8 7 6 5

Most effective for improving physical working capacity in laboratory animals

*

4 3 2 1 0

Control

Rhodiola

Rhodiola +H2O2

H2O2

May enhance the production of HSP70 (adaptogenic cellular protein) in muscles under conditions of increased oxidative stress

14 12 HSP70 (ng/mg protein)

Protected healthy volunteers from physical exercise induced muscle damage and inflammation

10 8 6

*

4 2 0

Control

Rhodiola

Rhodiola +H2O2

H2O2

Fig 3. // Effect of RhodioLife on (a) HSP70 gene expression levels and

(b) HSP70 protein concentration in H2O2-treated C2C12 muscle cells. Test groups were pre-treated treated with 10 ppm of RhodioLife for 1 hr followed by exposure to 2 mM H2O2 for 1 hr. HSP70 protein concentration was normalized by total protein. *p < 0.05, n=3 compared with control (Student麓s t-test).

RhodioLife 路 Product Developed & Manufactured by PoliNat | 35


Efficacy & Safety

SAFETY The Community monograph provided by the Herbal Medicinal Products Committee of the European Medicines Agency publicly available in 2012 reports in the overall conclusions on clinical safety of Rhodiola rosea that based on published data “… traditional herbal medicinal products containing Rhodiola rosea are not harmful when used in the specified conditions”. However said Committee points out the lack of experimental data on genotoxicity in the published literature (HMPC, 2012). A formal review of genotoxicity was performed on RhodioLife with the intent of achieving GRAS (Generally Regarded As Safe) status in the US. In the reverse mutation assay using bacteria performed under OECD 487 guidance, concentrations of 31.6 to 5000 µg/plate in experiments with and without metabolic activation did not cause gene mutations by base pair changes or frameshifts in the genome if the tester strains used (study number 120572). In the micronucleus test also performed in accordance to OECD, the test showed negative results (i.e. no abnormally increased number of multinucleated cells) at concentrations ranging from 80 to 620 µg/ml. At a concentration of 1250 µg/ml, the number of multinucleated cells was at least 2-fold higher than background levels, while higher concentrations were not evaluable due to cytotoxicity issues (yielding an insufficient umber of scorable cells). The test substance at 1250 µg/ ml also interfered with the assay detection method, making results obtained with concentrations of 1250 µg/ml and higher not conclusive due to solubility issues. An expert panel was later convened to evaluate all available data on the safety of Rhodiola rosea and particularly RhodioLife with the sole purpose of assessing its GRAS status under the Code of Federal Regulations. Their final position was that “the provided information combined with specific manufacturing information for RhodioLife, was sufficient to conclude that a single daily serving size of 50 mg would be considered safe when added to functional foods and beverages without

36 | RhodioLife · Product Developed & Manufactured by PoliNat

taking into account any allergic reactions or any potential interactions between RhodioLife and other consumed ingredients” (Pizzorno, 2012-in house report). Acute oral toxicity of RhodioLife has been tested in Sprague-Dawley rats following OECD 423, Directive 2004/73 B1 and OPTTS 870.1100 guidelines. The study consisted on initial dose-range finding study followed by a single oral dose of RhodioLife given by gavage at a level of 2000 mg/kg body weight (5M/5F). Animals were then followed for 14 days and later scarified for pathologic examinations. There were no deaths or clinical symptoms related to the treatment and no histopathology findings. The NOAEL was established at 2000 mg/kg (Abidov, 2006).

RHODIOLIFE SAFETY SUMMARY Reverse Mutation Assay No gene mutations The micronucleus test No abnormal increase of multinucleated cells Acute oral toxicity in rats No deaths NOAEL 2000 mg/kg

RHODIOLIFE REGULATORY STATUS US: Self-affirmed GRAS EU: Registered product (Nr. NUT/PL/AS code: PL1648/3)


About the Origin

REFERENCES

RhodioLife 路 Product Developed & Manufactured by PoliNat | 37


RhodioLife

PHYTOCHEMICAL COMPOSITION Avula B, Wang YH, Ali Z, Smillie TJ, Filion V, Cuerrier A,Arnason JT, Khan IA. RP-HPLC determination of phenylalkanoids and monoterpenoids in Rhodiola rosea and identification by LC-ESI-TOF. Bio Chrom 2009l; 23: 865-872. Brown, R.P., Gerbarg, P., Ramazanov, Z. Rhodiola rosea: a phytomedicinal overview. HerbalGram 2002; 56: 40-52. Furmanowa M, Skopinska RE, Rogala E, Hartwich M, Rhodiola rosea in vitro culture – phytochemical analysis and antioxidant action. Acta Soc Bot Pol, 1998; 67: 69-73. György Z Tolonen A, Neubauer P, Hohtola A. Enhanced biotransformation capacity of Rhodiola rosea callus cultures for glycosid production. Plant Cell Tiss Org 2005; 83: 129-35. György Z, Tolonen A, Pakonen M, Neubauer P, Hohtola A. Enhancing the production of cinnamyl glycosides in compact callus aggregate cultures of Rhodiola rosea by biotransformation of cinnamyl alcohol. Plant Sci 2004; 166(1): 229-36. Jiménez C, Riguera R. Phenylethanoid Glycosides in Plants : Structure and Biological Activity. Nat Prod Rep 1994; 11: 591-606. Kurkin VA, Z.G., Chemical composition and pharmacological properties of Rhodiola rosea. Khim Pharm Zurnal 1986; 20: 1231-44. Ma YC, Wang XQ, Hou FF, Ma J, Luo M, Lu S, Jin P, Terevsky N, Chen A, Xu I, Pate AV, Gorecki D. Rapid resolution liquid chromatography (RRLC) analysis for quality control of Rhodiola rosea roots and commercial standardized products. Nat Prod Commun 2011; 6: 645650. Markus G, Yurdanur Y and Khan IA. Analysis of the marker compounds of Rhodiola rosea L. (golden root) by reversed phase high performance liquid chromatography. Chem Pharm Bull 2001; 49(4): 465-7. Maslova LV, Kondratyev BY, Maslov LN and Lishmanov YB. On the cardioprotective and antiadrenergic activity of Rhodiola rosea extract during stress. Exp Clin Pharmacol 1994; 57: 61-63. Panossian A, Nikoyan N, Ohanyan N, Hovhannisyan A, Abrahamyan H, Gabrielyan E, Wikman G. Comparative study of Rhodiola preparations on behavioral despair of rats. Phytomed 2008; 15(1-2): 84-91. Panossian A, Wagner H. Stimulating effect of adaptogens: an overview with particular reference to their efficacy following single dose administration. Phytother Res 2005; 19(10): 819-38. Perfumi M, Mattioli L. Adaptogenic and central nervous system effects of single doses of 3% rosavin and 1% salidroside Rhodiola rosea L. extract in mice. Phytotherapy Res 2007; 21(1):37-43. Ramazanov Z, del Mar BS. In New Secrets of Effective Natural Stress and Weight Management Using Rhodiola Rosea and Rhododendron Caucasicum. ATN/Safe Goods Publishing: East Canaan 1999; 70-5.

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EFFICACY & SAFETY Abidov M, Crendal F, Grachev S, Seifulla R, Ziegenfuss T. Effect of extracts from Rhodiola rosea and Rhodiola crenulata (Crassulaceae) roots on ATP content in mitochondria of skeletal muscles. Bull Exp Biol Med. 2003 Dec;136(6):585-7. Abidov M, Grachev S, Seifulla RD, Ziegenfuss TN. Extract of Rhodiola rosea radix reduces the level of C-reactive protein and creatinine kinase in the blood. Bull Exp Biol Med. 2004 Jul;138(1):63-4. Abidov M. RhodioLife acute oral toxicity test in the rat. Project number: 2006/03-16 (in house report, available upon request under confidentiality agreement). Brekhman II, Dardymov IV. New substances of plant origin which increase nonspecific resistance. Ann Rev Pharmacol 1969;9:419-430. Evdokimov VG. Effect of cryopowder Rhodiola rosae L. on cardiorespiratory parameters and physical performance of humans. Aviakosm Ekolog Med 2009, 43(6):52-6. Furmanowa M, S.-R.E., Rogala E, Hartwich M., Rhodiola rosea in vitro culture – phytochemical analysis and antioxidant action. Acta Soc Bot Pol, 1998;67:69-73. Gorelick-Feldman J, Maclean D, Ilic N, Poulev A, Lila MA, Cheng D, Raskin I. Phytoecdysteroids increase protein synthesis in skeletal muscle cells. J Agric Food Chem. 2008 May 28;56(10):3532-7. doi: 10.1021/jf073059z. Epub 2008 Apr 30. HMPC. Assessment report on Rhodiola rosea L., rhizome et radix. EMA/HMPC/232100/2011. Published 27 March 2012. Hung SK, Perry R, Ernst E. The effectiveness and efficacy of Rhodiola rosea L.: a systematic review of randomized clinical trials. Phytomedicine. 2011 Feb 15;18(4):235-44. doi: 10.1016/j. phymed.2010.08.014. Epub 2010 Oct 30. Hung SK, Perry R, Ernst E. The effectiveness and efficacy of Rhodiola rosea L.: a systematic review of randomized clinical trials. Phytomedicine. 2011 Feb 15;18(4):235-44. doi: 10.1016/j. phymed.2010.08.014. Epub 2010 Oct 30. Ishaque S, Shamseer L, Bukutu C, Vohra S. Rhodiola rosea for physical and mental fatigue: a systematic review. BMC Complement Altern Med. 2012 May 29;12:70. doi: 10.1186/1472-6882-12-70. Kelly GS. Rhodiola rosea: A possible plant adaptogen. Altern Med Rev 2001;6(3):293-302. Khanum F, Singh AB, Brahm S. Rhodiola rosea: A versatile adaptogen. Comp Rev Food Sci Food Saf 4(3):55-62. Kurkin VA, Z.G., Chemical composition and pharmacological properties of Rhodiola rosea. Khim Pharm Zurnal, 1986;20:1231-1244 Ma, G., et al., Rhodiolosides A-E, monoterpene glycosides from Rhodiola rosea. Chem Pharm Bull (Tokyo), 2006;54:1229-33.

Sokolov S, Ivashin VM, Zapesochnaya G, Kurkin VA and Shchavlinskii AN. Studies of neurotropic activity of new compounds isolated from Rhodiola rosea. Khimiko-Farmatsevticheskii Zhurnal 1985; 19: 136771.

Morton JP, Kayani AC, McArdle A, Drust B. The exercise-induced stress response of skeletal muscle, with specific emphasis on humans. Sports Med. 2009;39(8):643-62. doi: 10.2165/00007256-200939080-00003.

Tolonen A, György Z, Jalonen J, Neubauer P, Hohtola A. LC/MS/MS identification of glycosides produced by biotransformation of cinnamyl alcohol in Rhodiola rosea compact callus aggregates. Biomed Chrom 2004; 18: 550-8.

Olsson EM, von Schéele B, Panossian AG. A randomised, double-blind, placebo-controlled, parallel-group study of the standardised extract shr5 of the roots of Rhodiola rosea in the treatment of subjects with stressrelated fatigue. Planta Med. 2009 Feb;75(2):105-12. doi: 10.1055/s0028-1088346. Epub 2008 Nov 18.

Tolonen A, Pakonen M, Hohtola A and Jalonen J. Phenylpropanoid glycosides from Rhodiola rosea. Chem Pharm Bull 2003; 51(4): 467-70. Van Diermen D, Marston A, Bravo J, Reist M, Carrupt PA, Hostettmann K. Monoamine oxidase inhibition by Rhodiola rosea L. roots. J Ethnopharmacol 2009; 122(2): 397-401.

38 | RhodioLife · Product Developed & Manufactured by PoliNat

Parisi A, Tranchita E, Duranti G, Ciminelli E, Quaranta F, Ceci R, Cerulli C, Borrione P, Sabatini S. Effects of chronic Rhodiola Rosea supplementation on sport performance and antioxidant capacity in trained male: preliminary results. J Sports Med Phys Fitness. 2010 Mar;50(1):57-63.


Pérez V, Hernández A, Jiménez-del-Rio M, Zubeldia JM.RhodioLife, a Rhodiola Rosea roots extract, modulates the expression of Heat Shock Protein 70 (HSP70): An in-vitro study. Poster presentation at Vitafoods, Geneve, Switzerland, May 2012. Pizzorno JE, Oktech HA, Oliff HS. Determination of the Generally Recognized As Safe (GRAS) Status of RhodioLife, (Rhodiola rosea Extract) as an Ingredient in Selected Foods. May 2012 (in house report, available upon request under confidentiality agreement). Skarpanska-Stejnborn A, Pilaczynska-Szczesniak L, Basta P, DeskurSmielecka E. The influence of supplementation with Rhodiola rosea L. extract on selected redox parameters in professional rowers. Int J Sport Nutr Exerc Metab. 2009 Apr;19(2):186-99. Study number 100000752. CEREP. ADME-Tox: In vitro micronucleus. Study of Rhodiola rosea extract (in house report, available upon request under confidentiality agreement). Study number 120572. BSL Bioservice. Reverse mutation assay using bacteria (Salmonella typhimurium and Escherichia coli) with Rhodiola rosea extract-5% rosavins (RhodioLife) (in house report, available upon request under confidentiality agreement).



RhodioLife | The Origin