Title Symphytum plants: A comprehensive review on chemical composition, food applications and phytopharmacology Articletype ReviewArticle Abstract… [623271]

Manuscript Details
Manuscript number TIFS_2019_10
Title Symphytum plants: A comprehensive review on chemical composition, food
applications and phytopharmacology
Articletype ReviewArticle
Abstract
Symphytum genus, commonly known as comfrey, is a member of Boraginaceae family that have been used both
internally and externally as an herbal medicine for more than 2,000 years for bone breakages, sprains and
rheumatism, liver problems, gastritis, ulcers, skin problems, joint pain and contusions, wounds, gout, hematomas, and
thrombophlebitis. Pharmacological effects of Symphytum extracts are attributed to several chemical compounds, such
asallantoin and phenolic compounds, including rosmarinic, p-hydroxybenzoic, caffeic, chlorogenic and p-coumaric
acids,glycopeptides, polysaccharides, and the toxic pyrrolizidine alkaloids. In light of Symphytum plants potential
indications, worldwide use and its capability for harm, it is desirable to explore the breadth of data available for all
indications, to identify coverage, gaps, areas of strong and weak research and the nature of adverse events. This help
thepractice of herbalists and healthcare practitioners by summarizing the amount and quality of evidence, often
scarcein herbal medicine, to support treatment of each indication with Symphytum plants and highlight any risk
associated with its usage. A focus on external use widens the applicability of this review to patients purchasing over-
the-counter preparations, as well as practitioners across many countries. Thus, the present review summarized data
onbothsize and nature of the evidence-based indications for Symphytum plants use, relating to both efficacy and
adverse events.
Keywords Symphytum plants, Traditional use, Phytochemistry, Biological activity, Clinical
trials
Manuscript region of origin MiddleEast
Corresponding Author JavadSharifi-Rad
Corresponding Author's
InstitutionPhytochemistry Research Center, Shahid Beheshti University of Medical
Sciences, Tehran, Iran
Orderof Authors BahareSalehi, Farukh Sharopov, Tugba Tumer, Adem Ozleyen, Celia
Rodriguez-Perez, Shahira Ezzat, Elena Azzini, Tahereh Hosseinabadi, Monica
Butnariu, Ioan Sarac, Cristian Bostan, Krishnendu Acharya, Surjit Sen, Mehdi
Sharifi-Rad, Kadriye Nur Kasapoglu, Ceren Dașkaya-Dikmen, Razieh Sharifi-
Rad,Beraat Özçelik, Navid Baghalpour, Javad Sharifi-Rad, Patrick Valere
TsouhFokou, Natália Martins
Suggested reviewers AntoniSureda, Abhay Mishra, Marcello Iriti, Manisha Nigam
Submission Files Included in this PDF
FileName [File Type]
coverletter 04.01.2019.doc [Cover Letter]
Research highlights_Symphytum-04.01.2019.docx [Highlights]
Abstarct 04.01.2019.docx [Abstract]
Symphytum-plants_04.01.2019.docx [Manuscript File]
Toviewall the submission files, including those not included in the PDF, click on the manuscript title on your EVISE
Homepage, then click 'Download zip file'.

Islamic Republic of Iran
Ministry of Health and Medical Education
Shahid Beheshti University of Medical Sciences, Tehran, Iran:DarDate: 04-01-2019
Editor-in-Chief
Trends in Food Science & Technology
We would like to submit the manuscript entitled
“Symphytum plants: A comprehensive review on chemical
composition, food applications and phytopharmacology” in the
hope that it can be considered for publication on Trends in Food
Science & Technology and of interest for the Journal readership.
The manuscript, including related data, figures and tables
has not been previously published and it is not under
consideration elsewhere. All authors have approved the
submitted version of the manuscript.
Sincerely yours
Javad Sharifi-Rad

1Research highlights
2
3Symphytum plants have a long history of use in traditional herbal medicine
4The most common Symphytum plants bioactive effects include as antioxidants and anti-
5inflammatory
6Phenolic compounds and alkaloids represent promising naturally-occurring chemical
7constituents in Symphytum plants
8Alkaloids have shown a broad-spectrum antimicrobial effect, being even interesting for
9food applications
10The topical of use of Symphytum plants for musculoskeletal problems has also been stated
11in clinical trials
12

Abstract
Symphytum genus, commonly known as comfrey, is a member of Boraginaceae family that have
been used both internally and externally as an herbal medicine for more than 2,000 years for
bone breakages, sprains and rheumatism, liver problems, gastritis, ulcers, skin problems, joint
pain and contusions, wounds, gout, hematomas, and thrombophlebitis. Pharmacological effects
of Symphytum extracts are attributed to several chemical compounds, such as allantoin and
phenolic compounds, including rosmarinic, p-hydroxybenzoic, caffeic, chlorogenic and p-
coumaric acids, glycopeptides, polysaccharides, and the toxic pyrrolizidine alkaloids. In light of
Symphytum plants potential indications, worldwide use and its capability for harm, it is desirable
to explore the breadth of data available for all indications, to identify coverage, gaps, areas of
strong and weak research and the nature of adverse events. This help the practice of herbalists
and healthcare practitioners by summarizing the amount and quality of evidence, often scarce in
herbal medicine, to support treatment of each indication with Symphytum plants and highlight
any risk associated with its usage. A focus on external use widens the applicability of this review
to patients purchasing over-the-counter preparations, as well as practitioners across many
countries. Thus, the present review summarized data on both size and nature of the evidence-
based indications for Symphytum plants use, relating to both efficacy and adverse events.

11Symphytum plants: A comprehensive review on chemical composition, food applications
2and phytopharmacology
3Bahare Salehi1, Farukh Sharopov2, Tugba Boyunegmez Tumer3, Adem Ozleyen4, Celia
4Rodríguez‐Pérez5, Shahira M. Ezzat 6,7, Elena Azzini8, Tahereh Hosseinabadi9, Monica
5Butnariu10, Ioan Sarac10, Cristian Bostan10, Krishnendu Acharya11, Surjit Sen11, 12, Mehdi Sharifi-
6Rad13,*, Kadriye Nur Kasapoglu14, Ceren Dașkaya-Dikmen14, 15, Razieh Sharifi-Rad16, Beraat
7Özçelik14,17, Navid Baghalpour18, Javad Sharifi-Rad16,19,**, Patrick Valere Tsouh Fokou20,***,
8Natália Martins 21,22, ****
9
101Student Research Committee, School of Medicine, Bam University of Medical Sciences, Bam,
11Iran
122Department of Pharmaceutical Technology, Avicenna Tajik State Medical University, Rudaki
13139, 734003, Dushanbe, Tajikistan
143Department of Molecular Biology and Genetics, Faculty of Arts and Science, Canakkale
15Onsekiz Mart University, Canakkale, 17020 Turkey
164Graduate Program of Biomolecular Sciences, Institute of Natural and Applied Sciences,
17Canakkale Onsekiz Mart University, Canakkale, 17020 Turkey
185University College Dublin (UCD) Institute of Food and Health, UCD, Belfield, Dublin 4,
19Ireland
206Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Kasr El-Ainy Street, Cairo
2111562, Egypt
227Department of Pharmacognosy, Faculty of Pharmacy, October University for Modern Science
23and Arts (MSA), 6thOctober, 12566 Egypt
248Centre for Research on Food and Nutrition, Council for Agricultural Research and Economics,
25Rome, Italy
269Department of Pharmacognosy and Biotechnology, School of Pharmacy, Shahid Beheshti
27University of Medical Sciences, Tehran, Iran
2810Banat’s University of Agricultural Sciences and Veterinary Medicine “King Michael I of
29Romania” from Timisoara, 300645, Calea Aradului 119, Timis, Romania
3011Molecular and Applied Mycology and Plant Pathology Laboratory, Department of Botany,
31University of Calcutta, Kolkata 700019, India
3212Department of Botany, Fakir Chand College, Diamond Harbour, West Bengal – 743331, India
3313Department of Medical Parasitology, Zabol University of Medical Sciences, Zabol, Iran
3414Istanbul Technical University, Chemical and Metallurgical Engineering Faculty, Food
35Engineering Department, Ayazağa Campus 34469 Maslak, Istanbul, Turkey
3615Istanbul Gedik University, Department of Gastronomy and Culinary Arts, 34876 Kartal,
37Istanbul, Turkey

23816Zabol Medicinal Plants Research Center, Zabol University of Medical Sciences, Zabol, Iran
3917Bioactive Research & Innovation Food Manufac. Indust. Trade Ltd., Katar Street, Teknokent
40ARI-3, B110, Sarıyer, 34467, Istanbul, Turkey
4118Phytochemistry Research Center, Shahid Beheshti University of Medical Sciences, Tehran,
42Iran
4319Department of Chemistry, Richardson College for the Environmental Science Complex, The
44University of Winnipeg, Winnipeg, MB R3B 2G3, Canada
4520Antimicrobial and Biocontrol Agents Unit, Department of Biochemistry, Faculty of Science,
46University of Yaounde 1, Ngoa Ekelle, Annex Fac. Sci, P.O. Box. 812, Yaounde-Cameroon
4721Faculty of Medicine, University of Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto,
48Portugal
4922Institute for Research and Innovation in Health (i3S), University of Porto, 4200-135 Porto,
50Portugal
51
52
53∗ Corresponding author. Department of Medical Parasitology, Zabol University of Medical
54Sciences, Zabol, Iran.
55∗∗ Corresponding author. Zabol Medicinal Plants Research Center, Zabol University of Medical
56Sciences, Zabol, Iran
57∗∗∗ Corresponding author. Antimicrobial and Biocontrol Agents Unit, Department of
58Biochemistry, Faculty of Science, University of Yaounde 1, Ngoa Ekelle, Annex Fac. Sci, P.O.
59Box. 812, Yaounde-Cameroon.
60∗∗∗∗Corresponding author. Faculty of Medicine, University of Porto, Alameda Prof. Hernâni
61Monteiro, 4200-319 Porto, Portugal.
62E-mail addresses: mehdi_sharifirad@yahoo.com (M. Sharifi-Rad), javad.sharifirad@gmail.com
63(J. Sharifi-Rad), tsouh80@yahoo.fr (P.V.T.F.), ncmartins@med.up.pt (N.M.).
64

365Abstract
66Symphytum genus, commonly known as comfrey, is a member of Boraginaceae family that have
67been used both internally and externally as an herbal medicine for more than 2,000 years for
68bone breakages, sprains and rheumatism, liver problems, gastritis, ulcers, skin problems, joint
69pain and contusions, wounds, gout, hematomas, and thrombophlebitis. Pharmacological effects
70of Symphytum extracts are attributed to several chemical compounds, such as allantoin and
71phenolic compounds, including rosmarinic, p-hydroxybenzoic, caffeic, chlorogenic and p-
72coumaric acids, glycopeptides, polysaccharides, and the toxic pyrrolizidine alkaloids. In light of
73Symphytum plants potential indications, worldwide use and its capability for harm, it is desirable
74to explore the breadth of data available for all indications, to identify coverage, gaps, areas of
75strong and weak research and the nature of adverse events. This help the practice of herbalists
76and healthcare practitioners by summarizing the amount and quality of evidence, often scarce in
77herbal medicine, to support treatment of each indication with Symphytum plants and highlight
78any risk associated with its usage. A focus on external use widens the applicability of this review
79to patients purchasing over-the-counter preparations, as well as practitioners across many
80countries. Thus, the present review summarized data on both size and nature of the evidence-
81based indications for Symphytum plants use, relating to both efficacy and adverse events.
82Keywords: Symphytum plants, Traditional use, Phytochemistry, Biological activity, Clinical
83trials.
84

4851. Introduction
86Symphytum genus, commonly known as comfrey, is a member of Boraginaceae family
87native to Europe and Western Asia, comprising approximately 35 species, including Symphytum
88officinale L., Symphytum tuberosum L., Symphytum x uplandicum Nyman, Symphytum asperum
89Lepech, and Symphytum caucasicum Bieb (Horinouchi & Otuki, 2013; Schmelzer & Gurib-
90Fakim, 2008). S. officinale, often called herb comfrey, is the most commonly used species and is
91a perennial plant with white, creamy yellow or pale purple flowers and long rough leaves (Frost,
92MacPherson, & O'Meara, 2013; C. Staiger, 2012). Other species that have been used in the past
93include Symphytum asperum (Prickly Comfrey) and Symphytum x uplandicum (Russian Comfrey
94— a hybrid of the latter two species (syn. Symphytum peregrinum) (Frost et al., 2013).
95Symphytum plants have been used both internally and externally as an herbal medicine for
96more than 2,000 years (Riet-Correa, Medeiros, Tokarnia, & Dobereiner, 2007). Worldwide,
97ethnographic studies report external application of S. officinale and S. tuberosum for bone
98breakages, sprains and rheumatism in Northern Navarra; a tea for liver problems, internal
99therapy for rheumatism in Mexico; gastritis and ulcers in a Brazilian marketplace; skin problems
100in the USA; the roots as a tea, alcoholic extract or ointment for bone and joint pain and
101contusions in Lithuania or as tonic in Jamaica (Cameron & Chrubasik, 2013). They are applied
102externally in the form of extracts, ointments, or compress pastes of Symphytum plants, and the
103leaf, herb or root has been used as traditionally to treat musculoskeletal disorders, wounds, joint
104inflammatory disorders, wounds, bone fractures, gout, hematomas, and thrombophlebitis (Frost
105et al., 2013; Riet-Correa et al., 2007; C. Staiger, 2012).
106The diversity of Symphytum bioactivities and related efficacies is a manifestation of its
107complex effects and provides a font of opportunity for researchers of various disciplines, i.e.

5108pharmacy (Frost et al., 2013). Pharmacological effects of Symphytum extracts are attributed to
109several chemical compounds. Allantoin has been claimed as the active ingredient of comfrey,
110which is responsible of cell division initiation and of growth of the conjunctive tissue, bones,
111cartilages and of acceleration of wound healing (MacKay & Miller, 2003; Elena Neagu, Paun, &
112Radu, 2010). S. officinale root has extract containing allantoin and phenolics including mainly
113rosmarinic, and p-hydroxybenzoic, caffeic, chlorogenic and p-coumaric acids has strong
114antioxidant potential and a beneficial effect on human skin fibroblasts (Ireneusz Sowa et al.,
1152018). Rosmarinic acid is an anti-inflammatory constituent; polyglucides an local soothing
116effect; and tannins, which are astringent. Glycopeptides isolated from the root have demonstrated
117anti-inflammatory activity in rat paw edema, and the crude juice of S. officinale leaves has
118increased the number of fibroblast and collagen fibers in rat lesions (Mazzocchi & Montanaro,
1192012). Barbakadze et al. indicated that leaves of Symphytum asperum and S. caucasicum had
120anticomplement and antioxidant activity (Vakhtang Barbakadze et al., 2011). Moreover, strong
121antioxidant activities have been attributed to the presence of polysaccharides having uronic acid
122group in comfrey root (Shang et al., 2018). Enzyme-ultrasonic assistance technology has been
123reported as a good way to extract polysaccharides from comfrey root that exhibit notable α-
124glucosidase inhibition activity (Chen, Shang, Yang, Li, & Wu, 2018).
125Despite its folkloric uses, Symphytum plants primarily contains symphytine and
126echimidine, pyrrolizidine alkaloids, which have been linked to hepatic cancers and
127hepatotoxicity, pneumotoxicity, genotoxicity and carcinogenicity in chronic animal and clinical
128studies (Adeneye, 2014; Brown et al., 2016; Kruse, Stegemann, Sievert, & Ober, 2017; Kurucu,
129Kartal, Choudary, & Topcu, 2002; Mei et al., 2010). In humans the consumption of Symphytum
130plants has been associated with individual cases of hepatotoxic reactions: liver fibrosis, portal

6131hypertension, and veno-occlusive disease (Mazzocchi & Montanaro, 2012). Conversely, no
132adverse reactions have been reported in association with external use and pharmacokinetic
133studies suggest that cutaneous absorption is very low (Kruse et al., 2017). Hence, its internal
134application is not recommended by regulatory agencies and international health organizations
135(Brown et al., 2016). Nevertheless, as a precaution, in the UK, Symphytum plants is available
136over-the-counter in external preparations only, and for internal or external use when prescribed
137by qualified medical herbalists. In USA, Canada and some European countries such as Germany,
138Denmark and Austria the Symphytum species is under restriction. Commission E recommends
139restricting its use to 4-6 weeks per year (Colegate, Stegelmeier, & Edgar, 2012).
140S. officinale leaf extract loaded silver nanoparticles has been suggested to be used as an
141agent against skin photoaging due to its photoprotective potential (Singh, Du, Singh, & Yi,
1422018). Among the chemical compounds of comfrey, rosmarinic acid showed antiviral,
143antioxidant, antimutagenic and anticancer activities (Amakura, Okada, Tsuji, & Tonogai, 2000;
144Bala, Bhardwaj, Hariharan, & Kumar, 2006). Another important bioactive compound of comfrey
145is considered as ellagic acid (Vesna Lj Savić et al., 2015). Ellagic acid could be effective on the
146reduction of heart disease, birth defects, liver problems, the side effects of chemotherapy in
147patients with advanced prostate cancer. It has also antioxidative activity (Häkkinen, Kärenlampi,
148Mykkänen, Heinonen, & Törrönen, 2000).
149Extracts from comfrey leaves showed inhibition of fungal pathogens germination and
150modulate plant defense mechanisms (Karavaev et al., 2001b). The studies on comfrey extracts
151reported that this plant could be used in the management of pests and diseases (Pileggi, Raiman,
152Micheli, Beatriz, & Bobalto, 2002). In addition to antifungal activity of comfrey, Avancini et al.
153showed the antibacterial activity of S. officinale against the bacteria causing bovine mastitis

7154(Avancini, Wiest, Dall’Agnol, Haas, & von POSER, 2008). Indeed, Oliveira et al. have used the
155comfrey in the broiler diet and found that S. officinale could be used as a growth-promoting
156antibiotic for feeding broiler (Rodrigues Oliveira et al., 2016).
157Clinical trials have examined comfrey for back pain, wounds and arthritis, but the full
158scope of evaluative research has not been previously determined. Its pharmacological properties
159suggested that comfrey may be an alternative solution for treating skin conditions (Horinouchi &
160Otuki, 2013). A review of herbal therapies for osteoarthritis found moderate evidence for the
161effectiveness of Symphytum plants cream (Cameron & Chrubasik, 2013). At the same time,
162serious adverse events were not reported in trials or observational studies. However, the safety
163assessments were mostly performed in individual case studies and obtained from animal data
164using excessively high doses. There is a lack of safety assessment using bio-chemical markers
165and epidemiological methods in humans (Frost et al., 2013).
166The aim of this manuscript is to review the recent studies on Symphytum plants including
167their habitat and cultivation conditions, phytochemical composition, and to discuss their
168antimicrobial, antioxidant, anticancer activities by in vitro and in vivo studies and their clinical
169effectiveness.
1702. Habitat and cultivation of Symphytum plants
1712.1. Habitat
172Symphytum genus is a mesophytic, perennial herb belonging to family Boraginaceae. The
173genus comprises approximately 40 species, among them 14 species are reported from Europe
174mainly in peninsular Italy (Pawlowski, 1972; Peruzzi, Garbari, & Bottega, 2001) and around 18 –
175species are reported from Turkey (Tarıkahya, 2010). Symphytum spp. are native to Europe and

8176Asia with a centre of origin in the Pontic province of the Euro-Siberian region (Hacıoğlu & Erik,
1772011). It thrives well in moist cool places on river banks and streams, diches of roads and damp
178grasslands.
179
1802.2. Cultivation
181Optimum growing parameters
182Symphytum spp. grows in temperate and subtropical regions. It adapted well and gives
183maximum yield under cold condition with full sunlight (Teynor et al., 1992). Due to its deep root
184system the plant is drought tolerant. The plant is also very frost resistant. It is a fast-growing
185plant. If weeds and soil fertility is properly maintained the crop should last indefinitely.
186
187Soil
188Symphytum spp. is widely adaptable to many soils but prefers moist, fertile and rich in
189organic matter (Chittendon, 1956; Grieve, 1984). The plant grows well in sandy looms with
190adequate nutrients mainly if nitrogen is present in the soil (Teynor et al., 1992). The ideal soil pH
191ranges from 6.0 to 7.0. Huxley reported that comfrey thrive better in an open sunny site in a deep
192rich soil if it is amended with compost material (Huxley, 1992).
193
194Propagation
195The crop is generally propagated through transplants, crown cuttings with buds and root
196cuttings as rate of seed production is very low (Bogert, Briggs, & Calloway, 1973). High yield is
197recorded from transplant and least from root cutting during the first year of propagation but after
198that there is no difference in yield regardless of propagation method. Propagation through root

9199cuttings are less expensive and frequently used method. Cutting size varies from 1½ to 6 inches
200long and from ¼ to ¾ inches in diameter are common. Root cutting with comparatively smaller
201size generate plants but longer cutting establish and emerge faster. It has been reported that
202successful in vitro propagation from root explants produces large number of clones in a short
203time than by the conventional vegetative propagation (Harris, Grove, & Havard, 1989).
204
205Planting
206The best time of comfrey planting is April, but the crop can be planted throughout the
207growing season. Teynor et al. reported that root cutting should be planted before September,
208while early October is suitable for both transplant or crown cuttings (Teynor et al., 1992).
209Cuttings should be treated with cold water for adequate time before planting (Robinson, 1983).
210Planting depth varies with soil texture and soil moisture content. Four inches depth is common
211but up to 2 inches depth is also practiced with adequate irrigation system. Symphytum spp. is
212generally planted in a checkerboard pattern at rows of 3 to 4 ft apart to allow cross cultivation for
213weed control. It has been found that closer row spacing (about 30 inches) gives better yield but
214the cost of cutting become higher (Robinson, 1983; Teynor et al., 1992).
215
216Maturing
217Symphytum spp. contains high amount of proteins and the plant obtains all its nitrogen
218from the soil. So, it is necessary to amend nitrogen fertilizer or compost by broadcasting or side
219dressing (Thoresen, 2013). Older plantings that develop lighter green color leaves require
220broadcasting of nitrogen fertilizer. Barnyard manure is an excellent fertilizer for Symphytum.
221Sometime powdered limestone and rock phosphate can be added if the soil is too acidic. The

10222crop can be benefited from the addition of animal manure or poultry litters as a mulch or can also
223be mulched with other nitrogen rich materials such as lawn clippings.
224
225Weed and pest management
226Due to fast and dance growth habit of comfrey it is an excellent weed competitor. Weeds
227may establish between the clumps of Symphytum spp. under a multiple-cut harvesting practice.
228Rototilling between plants is an effective method of controlling weeds (Teynor et al., 1992). The
229crop usually been grown without herbicides. It has been reported that Symphytum spp. are
230tolerant to 2,4-D and 2,4,5-T and susceptible to atrazine, sodium chloride and ammonium
231sulfamate (Robinson, 1983). Diseases have not been a serious problem in this crop. It is reported
232that rust fungus (Melampsorella symphyti) is associated with the roots which reduces the yield
233(Teynor et al., 1992). A rare association of alfalfa mosaic virus (AMV) was reported in
234Symphytum spp., which showed yellow spots, rings and chlorotic line patterns (Bellardi & Benni,
2352005). Insect pest is also not reported much in this crop. Foliar nematodes (Aphelenchoides
236fragariae and A. ritzemabosi) are associated with Symphytum spp. causes reduction in crop yield
237(Juhl, 1978; Knight, Barber, & Page, 1997).
238
239Harvesting
240Mature Symphytum spp. can be harvested 4-5 times/year. When the seasonal conditions
241are suitable, usually during mid-spring and the crop reaches a height of about 24 inches they are
242ready for harvesting. Best time to cut the crop is just before flowering as it is the most effective
243in terms of the nutrient that it produces (Robinson, 1983).

112443. Phytochemical composition of Symphytum plants
245Several therapeutic properties attributed to Symphytum plants came from different main
246constituents present in root together with the leaves and the flowering tops of the plants. Their
247therapeutic properties are based on its anti-inflammatory, analgesic, granulation promoting, and
248anti-exudative properties, because comfrey contains varying quantities of allantoin, rosmarinic
249acid, triterpene saponins, tannins, alkaloids, amino acids, flavonoids, triterpenes, terpenoids,
250tannins, saponins, sterols, mucopolysaccharides, and other hydroxycinnamic acid derivatives,
251which are critically important for its pharmacodynamic characteristics (E. Neagu, Paun, &
252Radu, 2011 ; Stickel & Seitz, 2000). Symphytum species contain a variety of constituents,
253including carbohydrates, tannins, amino acids, allantoin, polysaccharides, triterpenes, sterols,
254phenolic compounds, and pyrrolizidine alkaloids (Mulkijanyan et al., 2009; Elena NEAGU,
255PĂUN, & RADU, 2011; Trifan et al., 2018) that are summarized in Table 1. Chemical structure
256of the main heterocyclic compounds of the Symphytum plants presented in Figure 1.
257
2583.1. Alkaloids
259Many pyrrolizidine alkaloids known for their high toxicity and biological activity have
260been isolated and characterized from Symphytum genus (Mulkijanyan et al., 2009; Tamariz,
261Burgueño-Tapia, Vázquez, & Delgado, 2018). Lasiocarpine, echimidine and symphytine
262reported as most toxic pyrrolizidine alkaloids in Symphytum plants (Onduso, 2014). The use of
263comfrey leaves recognized as a substantial health hazard with hepatic toxicity in humans and
264carcinogenic potential in rodents, most likely, due to various hepatotoxic pyrrolizidine alkaloids
265such as symviridine, symlandine, asperumine, lasiocarpine, symphytine and their related N-
266oxides (Melkumova, Telezhenetskaya, Yunusov, & Manko, 1974; E. Roeder, 1995; l. E. Roeder,

12267Bourauel, & Neuberger, 1992; Stickel & Seitz, 2000). As well, 6,7-dihydro-7-hydroxy-1-
268hydroxymethyl-5H-pyrrolizine, the metabolic product of a series of tumorigenic pyrrolizidine
269alkaloids in the liver of rats (Chou & Fu, 2006) was linked to its toxic effects (Gomes, Massoco,
270Xavier, & Bonamin, 2010).
271Alkaloids from Symphytum species have been extensively investigated both
272quantitatively and qualitatively, and identified echimidine, echimidine N-oxide, intermedine N-
273oxides, intermedine,7-acetyl intermedine, 7-acetyl lycopsamine, lycopsamine, lycopsamine N-
274oxides, symphytine N-oxide, symphytine, symviridine and symviridine N-oxide as major
275constituents.
2763.1.1. Symphytum asperum Lepech
277Pyrrolizidine alkaloids, echimidine, lycopsamine N-oxides, 7-acetyl lycopsamine, 3-
278acetyllycopsamine, intermedine N-oxides, symphytine, 7-acetyl symlandine, symviridine, 7-
279acetyl symviridine, myoscorpine, triangularine and heliosupine were detected in the root samples
280of S. asperum Lepech (Onduso, 2014; E. Roeder, 1995; l. E. Roeder et al., 1992).
2813.1.2. Symphytum caucasicum Bieb.
282Asperumine, echimidine N-oxide, echinatine, and lasiocarpine were the major alkaloids
283found in S. caucasicum roots growing in Uzbekistan (Melkumova et al., 1974; E. Roeder, 1995).
2843.1.3. Symphytum cordatum (L.) W.K.
285Eighteen various alkaloids: echimidine N-oxide (three diasteroisomers), 7-sarracinyl-9-
286viridiflorylretronecine (two diasteroisomers), echimidine (two diasteroisomers), lycopsamine
287(two diasteroisomers), dihydroechinatine N-oxide, dihydroheliospathuline N-oxide, lycopsamine

13288N-oxide (three diasteroisomers), 7-acetyl lycopsamine-N-oxide, symphytine N-oxide (two
289diasteroisomers) and 2,3-epoxyechiumine N-oxide have been tentatively determined for the first
290time in the alkalized crude extract of S. cordatum (L.) W.K. roots (Mroczek, Ndjoko-Ioset,
291Głowniak, Mietkiewicz-Capała, & Hostettmann, 2006).
2923.1.4. Symphytum officinale L.
293In 1993, Mutterlein and Arnold reported no alkaloid-free root in more than 300 samples
294of S. officinale plants coming from over 150 different natural habitats (Mutterlein & Arnorld,
2951993). Pyrrolizidine alkaloids (intermedine, acethylintermedine, lycopsamine, echimidine,
296symviridine) contents ranged between 0.04 – 0.6 % (depending on plant part) in S. officinale
297(Stickel & Seitz, 2000). Pyrrolizidine alkaloids are distributed not uniformly within the plants,
298their concentrations of the roots were a 100-fold higher than the aerial parts (Mutterlein &
299Arnorld, 1993). The total pyrrolizidine alkaloid contents ranged 1380- 8320 μg/g root and 15-55
300μg/g leaf of common comfrey, respectively (C.E. Couet, C. Crews, & A.B. Hanley, 1996). The
301two major pyrrolizidine alkaloids were symphytine and echimidine, they varied considerably
302between teas prepared from comfrey leaves purchased from the different vendors of plant
303material (Oberlies et al., 2004 ). Content (ng/g) of pyrrolizidine alkaloids from different parts of
304S. officinale L. represented in Table 2.
305Twenty three alkaloids: intermedine, lycopsamine, intermedine N-oxide, lycopsamine N-
306oxide, 7-acetylintermedine, 7-acetyllycopsamine, 7-acetylintermedinenoxide, 7-
307acetyllycopsaminenoxide, uplandicine N-oxide, myoscorpine, echiumine, symphytine,
308symviridine, myoscorpine N-oxide, echiumine N-oxide, symphytine N-oxide, symviridine N-
309oxide, heliosupine, asperumine, heliosupine N-oxide, asperumine N-oxide were identified from

14310the roots of S. officinale (Avula et al., 2015). Pyrrolizidine alkaloid lycopsamine, echimidine,
311echimidine N-oxide, symviridine, symlandine, symphytine, symphytine N-oxide, intermedine,
312intermedine N-oxide, acetyl intermedine, acetyl lycopsamine, acetyl intermedine N-oxide, acetyl
313lycopsamine N-oxide, lasiocarpine, uplandicine, uplandicine N-oxide were reported in the roots
314of S. officinale (Altamirano, Gratz, & Wolnik, 2005; Brauchli, Lüthy, Zweifel, & Schlatter,
3151982; Janes, Kalamar, & Kreft, 2012 ; Janeš & Kreft, 2014 ; Kim et al., 2001; F. Liu et al., 2009;
316Tsutomu Furuya & Hikichi, 1971).
3173.1.5. Symphytum tuberosum
318Anadoline and echimidine alkaloids were isolated from whole plant extract of S.
319tuberosum originated in Turkey (Ulubelen & Öcal, 1977).
3203.1.6. Symphytum x uplandicum Nyman (syn. S. peregrinum Ledeb.)
321Pyrrolizidine alkaloids, symviridine (l. E. Roeder et al., 1992), intermedine, intermedine
322N-oxide, 7-acetyl intermedine, echimidine, echimidine N-oxide, lycopsamine, 7-acetyl
323lycopsamine, acetyl lycopsamine N-oxide, symphytine, symphytine N-oxide, symlandine,
324myoscorpine, uplandicine, uplandicine N-oxide have been identified in the leaves and roots of S.
325x uplandicum (Altamirano et al., 2005; Culvenor, Edgar, Frahn, & Smith, 1980 ; E. Roeder,
3261995).
3273.2. Allantoin
328Allantoin was identified in shoot and root extracts of S. officinale and S. cordatum
329species (Dresler, Szymczak, & Wojcik, 2017). It was found in roots (E. Neagu et al., 2011 ; V.L.

15330Savić et al., 2015) and seeds of S. officinale (Al-Nimer & Wahbee, 2017). Allantoin is a
331metabolic compound of uric acid oxidation products. Natural allantoin is safe, non-toxic,
332compatible with cosmetic raw materials for its soothing, skin softening and healing activity
333(Becker et al., 2010).
3343.3. Phenolic constituents
335Trifan et al reported Symphytum root as an important source of phenolic compounds such
336as rosmarinic acid and salvianolic acids (A, B and C) endowed with antioxidant activity
337contributing positively to the overall bioactivity of Symphytum-derived preparations (Trifan et
338al., 2018). The presence of twenty phenolic acids including caffeic, p-coumaric and m-
339hydroxybenzoic acids were found in herb and roots of S. officinale by GC and HPLC (Grabias &
340Swiatek, 1998). p-Hydroxybenzoic, caffeic, rosmarinic, and chlorogenic acids were identified in
341shoot and root extracts of S. officinale and S. cordatum species (Dresler et al., 2017).
3423.4. Saccharides
343A new anti-complementary dihydroxycinnamate-derived polymer was isolated from the
344S. asperum roots (V.V. Barbakadze, Kemertelidze, Shashkov, & Usov, 2000). Poly[oxy-1-
345carboxy-2-(3,4-dihydroxyphenyl) ethylene] was the principal component in four water-soluble
346high-molecular preparations from the roots and stems of S. asperum and S. caucasicum. A
347biologically active polymer, poly[3-(3,4-dihydroxyphenyl)glyceric acid] were isolated from the
348roots of S. asperum and S. caucasicum (V. Barbakadze et al., 2005). 3-O-[beta-D-
349glucopyranosyl-(1->4)-beta-D-glucopyranosyl-(1->4)-alpha-L – arabinopyranosyl]-hederagenin-
35028-O-[alpha-L-rhamnopyranosyl- (1->4)-beta-D- glucopyranosyl-(1->6)-beta-D-glucopyranosyl]

16351ester, a bidesmosidic hederagenin hexasaccharide was isolated from the roots of S. officinale
352(Mohammad, Noorwala, Ahmad, & Sener, 1995 ). The fructan synthesizing capacity of the
353different calli and the regenerated plants were analyzed. Calli derived from ovaries, anthers, and
354roots, which are known to contain large amounts of fructan, were not capable of synthesizing
355fructan. Uniquely, calli derived from the leaves of the original plant synthesized fructan (Abou-
356Mandour, Czygan, Haaß, & Franz, 1987).
3573.5. Terpenoids
358Symphytoxide A, a triterpenoid saponin has been isolated from the ethanolic extract of
359the roots of S. officinale (Ahmad, Noorwala, Mohammad, Sener, & Aftab, 1993).
3603.6. Fatty acid and sterols
361The greatest amounts of γ-linolenic acid (16-72 %) was found in polyunsaturated fatty
362acids fraction from seeds of S. officinale (Yunusova, Lyashenko, Fedorov, Fedorov, &
363Denisenko, 2017). Isobauerenol, β-sitosterol have been found in the roots of S. officinale
364(Tsutomu Furuya & Hikichi, 1971).
365Overall, pyrrolizidine alkaloids were most investigated phytochemicals in the root of S.
366asperum Lepech, S. caucasicum Bieb., S. cordatum (L.) W.K., S. officinale L., S. tuberosum and
367S. x uplandicum Nyman. Numbers of biologically active polymers were found from the roots and
368stems of S. asperum and S. caucasicum. Beside alkaloids and saccharides, many common
369phenolic compounds were identified in Symphytum plants.
370
3714. Traditional use of Symphytum plants

17372The genus Symphytum is native to Western Asia and Europe, and has about 35 species
373(Üstün Alkan, Anlas, Ustuner, Bakırel, & Sari, 2014). Symphytum folk names include boneset,
374knitbone and its Latin name Symphytum derived from the Greek symphis, meaning growing
375together of bones, and phyton, a plant, referring to its ancient uses. Comfrey's main name,
376knitbone derives from the external use for improving sprains, burning and bruising. S. officinale
377has been used both internally and externally as a herbal remedy for more than 2,000 years in
378many countries (Rode, 2002). Symphytum extracts have been used in folk and traditional
379medicine for healing of various diseases (Merlani et al., 2010). The oldest use of Symphytum was
380probably topical. For example, Comfrey was a medication for fractures, dislocations, contusions,
381in ancient and medieval times (Kucera, Kálal, & Polesna, 2000). The farmers prepared ointments
382and tinctures from comfrey (S. officinale, Symphyti radix, Symphyti herba) roots, or used its
383leaves directly to treat sprains, contusions, swollen joints, or indigestion (Schmid et al., 2012). S.
384officinale is used in Romanian traditional medicine to treat different human and animal disease
385(Elena Neagu, Moroeanu, & Radu, 2008). S. officinale is applied externally in the form of
386extracts, ointments, or compress pastes of comfrey leaves and roots to treat joint disorders,
387bruises, sprains, pulled muscles and ligaments, hemorrhoids, bone fractures, tendon damage,
388ulcerations in the gastrointestinal tract, lung congestion, joint inflammation, and to promote
389wound healing (Frost et al., 2013; Rode, 2002), inflammatory disorders of wounds, joints,
390distortions, hematomas, and thrombophlebitis (Riet-Correa et al., 2007). As infusions and
391extracts, these plants are used for internal applications in treating gastritis, gastroduodenal ulcers,
392and lung congestion (Alkan, Anlas, Ustuner, Bakırel, & Sari, 2014; Frost et al., 2013; C. Staiger,
3932013). In German system of medicine, S. officinale has been used against skeletal muscle disease
394(Alkan et al., 2014; Kucera et al., 2000) while in Brazil, its infusion is used for liver problems,

18395gastritis and ulcers (de Albuquerque, Monteiro, Ramos, & de Amorim, 2007). In Mexico and
396Turkey aerial parts are used for native treatment of rheumatism (Aceves-Avila, Medina, & Fraga,
3972001) and gastrointestinal ulcers, respectively (Alkan et al., 2014). In the USA, it is used for skin
398problems (N. Smith, Shin, Brauer, Mao, & Gelfand, 2009). Its roots are used as a tea or ointment
399for bone pain and contusions in Lithuania (Petkeviciute et al., 2010). External application of root
400of S. officinale and S. tuberosum based-clay balms for bone breakages, sprains and rheumatism
401was reported in Northern Navarra (Cavero, Akerreta, & Calvo, 2011; Cavero & Calvo, 2015).
402
4035. Biological activity of Symphytum plants
404There is a great interest in searching of new food preservative and antimicrobial agents
405primarily among plant extracts with aim to discover new chemical structures for counteracting
406food spoilage and microbial resistance(M. Sharifi-Rad, J. Nazaruk, et al., 2018). The widespread
407uses of antibiotics in clinical medicine, agriculture, and veterinary increasing the development of
408antibiotic resistances among infectious microbial strains have impaired the treatment of
409pathogenic microbes (Kapil, 2005; Lewis & Ausubel, 2006; Matthew & Leach, 2004; M. Sharifi-
410Rad, P. Fokou, et al., 2018) indicated numerous herbs and their extracts how potentially
411favorable to wound healing, including the ability in acting as an antimicrobial, antifungal,
412astringent and so on. Koll R. et al attributed the therapeutic properties of S. officinale mostly to
413the anti-inflammatory, analgesic and anti-exudative properties as well as the activity in
414stimulating granulation and tissue regeneration (Koll et al., 2004).
415

194165.1 Potential application as food preservatives
417 Food preservation is made to preserve the natural characteristics and appearance of foods
418and to increase the shelf value of food for storage. The importance of food preservation mainly
419resides in the role that foodborne disease plays in human health and well-being(Salehi et al.,
4202018). According to the World Health Organization (WHO), 31 global hazards including virus,
421bacteria, protozoa, helminths and chemicals, caused 600 million foodborne illnesses and 420,000
422deaths worldwide in 2010 (WHO, 2015).
423Nowadays, conventional preservatives are synthetic chemical substances which include
424some that are being re-evaluated e.g. nitrates and nitrites by the corresponding authority due to
425their potential health risk (EFSA, 2015a). In fact, over 60% of the population recognized to be
426worry about the presence of food additives, including food preservatives in foods in 2010 (T. r.-
427E. v. o. E. s. f. a. A. i. S. EFSA, 2018. https://www.efsa.europa.eu/en/press/news/120130b 2012).
428For that reason, new natural compounds are being investigated to be used as food preservatives.
429These new trend is called biopreservation which aims the enhancement of food shelf life and
430food safety using plants, animals, microorganisms, and/or their metabolites(Lee & Paik, 2016;
431Settanni & Corsetti, 2008). In fact, traditionally, many plants-derived preservatives e.g. vinegar,
432citrus fruit juices and some plant extracts have been employed to preserve foods for centuries
433(MacDonald & Reitmeier, 2017).
434The access to these natural food preservatives increases in importance in developing
435countries where the resources to preserve foods are scarce. Their protective effect has been
436associated with their composition in bioactive compounds such as phytochemicals which protect
437them against viruses, bacteria or fungi. Despite Symphytum species and especially S. officinalis
438have been commonly employed as anti-inflammatory topically (C. Staiger, 2012), there is a

20439scarcity of information regarding the use of Symphytum species for food preservation. However,
440due to its chemical composition which includes allantoin, polysaccharides, glycopeptides, amino
441acids, triterpene saponins, alkaloids or phenolic compounds, among others, they should not be
442discarded as potential food preservatives. The most known preservatives in foods are
443antimicrobial and antioxidants agents. In this regard, Table 3 shows the antimicrobial, antifungal
444and antioxidant activities of Symphytum species.
445Food antioxidants are mainly employed to protect food from oxidants which can alter
446their organoleptic, texture and safety properties (Carocho, Morales, & Ferreira, 2018). For that
447purpose, the European Food Safety Authority (EFSA) has approved several food antioxidants
448including ascorbates, tocopherols, gallates, erythrobates, butylates, lactates or rosemary (Carocho
449et al., 2018). Among the Symphytum species, one of the most studied is S. officinale.
450Specifically, S. officinale roots have demonstrated to be a good source of phenolic compounds
451(V. L. Savić et al., 2015). Sowa et al. also found five phenolic acids in a S. officinale root extract,
452including rosmarinic acid, which showed a high in vitro antioxidant activity (I. Sowa et al.,
4532018). In agreement, Trifan et al. quantified several phenolic compounds in a comfrey root
454extracts including rosmarinic acid as the major one followed by salvianolic acids isomers.
455Rosmarinic acid has been highlighted as the most abundant with about 8 mg g-1 extract (Trifan et
456al., 2018). In the same work they showed the higher antioxidant activity of a comfrey root extract
457compared to standard antioxidants through different methods, including 2,2'-azino-bis(3-
458ethylbenzothiazoline-6-sulphonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH),
459reducing power assay and 15-lipoxygenase (15-LO) inhibition assay, thus, suggesting that
460phenolic compounds may play a major role in antioxidant activities of comfrey root extract
461(Trifan et al., 2018). In agreement with the aforementioned results, Neagu et al. described higher

21462antioxidant activity of comfrey extracts processed by ultrafiltration than ascorbic acid (E. Neagu
463et al., 2011 ), a common additive already used as food preservative (E-300)(EFSA, 2015b).
464Within the food preservatives employed as antimicrobials are found sodium benzoate,
465calcium propionate, and potassium sorbate, among others (MacDonald & Reitmeier, 2017;
466Wedzicha, 2003). On the other hand, the most recognized natural plant-derived antimicrobial
467compounds encompass phenolic compounds, essential oils or antimicrobial peptides (Lee &
468Paik, 2016; Sharifi-Rad et al., 2016). Nevertheless, alkaloids, natural occurring organic
469compounds in plants, have shown to have a broad spectrum antibacterial and antifungal activity
470(Erdemoglu, Ozkan, & Tosun, 2007; Morteza-Semnani, Amin, Shidfar, Hadizadeh, & Shafiee,
4712003; Slobodníková, KoSt'álová, Labudová, Kotulová, & Kettmann, 2004). In this regard,
472alkaloids represent another group of important compounds described in Symphytum species
473(Wuilloud, Gratz, Gamble, & Wolnik, 2004). They are more concentrated in leaves than in other
474parts of the plant and precisely comfrey leaves have been traditionally consumed as tea (Oberlies
475et al., 2004 ). However, several studies showed that pyrrolizidine alkaloids could be hepatotoxic
476(Ma et al., 2018; Neuman, Cohen, Opris, Nanau, & Jeong, 2015). They are not exclusively
477present in Symphytum species since they can be found in more than 300 plant species (Ma et al.,
4782018; Stickel & Seitz, 2000). Interestingly, earlier studies have demonstrated that alkaloids
479isolated from Symphytum sylvaticum leaves and roots are effective against several fungal cultures
480in vitro, with the root alkaloid extract rich in echimidine-N-oxide being more protective against
481certain fungi than leaves (Kartal, Kurucu, & Choudary, 2001). In addition, early studies carried
482out with comfrey leaves with high a content of phenolic compounds calculated per unit of
483biomass than the leaves, demonstrated to be effective against the germination of conidia and
484uredospore of pathogenic fungi when the aqueous extract was spraying wheat seedlings

22485(Karavaev et al., 2001a). In contrast, Savić et al. did not find any activity of a comfrey root
486aqueous extract against the fungi Aspergillus niger and Candida albicans (V. L. Savić et al.,
4872015).
488Little is known about the antibacterial effect of Symphytum species. In this context,
489Sumathi et al. tested the effect of different comfrey leaves extracts against several pathogenic
490strains i.e. Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Salmonella typhi,
491Escherichia coli and Proteus vulgaris. They found that comfrey leaves extract exhibited partial
492and strong inhibition against S. aureus, B. subtilis, P. aeruginosa, S. typhi (Sumathi, Kumar, Bai,
493& Glory, 2011). Though authors did not attribute its effect to a specific group of compound, the
494antimicrobial mechanism of action of comfrey leaves might be due to its composition in phenolic
495compounds which could act by interfering with bacterial cell permeability, binding to adhesins or
496inhibiting the DNA or RNA replication and transcription (Lee & Paik, 2016). Whilst comfrey
497leaves also showed inhibitory effect against Bacillus cereus, their roots extract presented a
498maximum inhibitory effect against Proteus vulgaris and S. aureus (Sumathi, 2016). In addition,
499Escherichia coli ATCC8739 and Salmonela typhimirium ATCC6538 were highly sensitive to an
500aqueous extract of comfrey roots (Vesna Lj Savić et al., 2015). Contrarily to the above-
501mentioned results, Woods-Panzaru et al. did not find any antimicrobial activity against 34
502microorganisms, including 24 bacteria and 10 fungi of 50 mg/mL of comfrey leaf extract
503(Woods-Panzaru et al., 2009). Thus, the lack of conclusive studies and the studies mainly
504focused on food microbial and fungal makes necessary more studies to determine the real
505antifungal and antibacterial effect of comfrey roots and leaves. Despite the lack of compositional
506comprehensive studies, preliminary data suggests that they are a valuable source of potentially
507bioactive compounds which could be used as food preservatives (Table 3). Nevertheless, more

23508studies are necessaries to elucidate the full chemical composition of different Symphytum
509species, their bioactivity as well as their toxicity.
510
5115.2. Antimicrobial activities of Symphytum plants
512There is the increasing use of a traditional medicine as an alternative form for treating
513various diseases due to the resistance of microorganisms to the existing synthetic antibiotics as
514well as alternative to discover new substances (Abdolshahi et al., 2018; A. M. Prakash et al.,
5152018; Prakash Mishra et al., 2018; J. Sharifi-Rad, G. Tayeboon, et al., 2018). In several
516countries, it is common to use plants and their parts in disease treatment and WHO (2014)
517reported the important role of traditional medicine highlighting different forms and specific types
518of this medicine (WHO, 2014). The genus Symphytum plants are well recognized as useful for
519medicinal preparations, due to their healing and therapeutic properties. The present section
520assessed the possible antimicrobial activities of these plants by in vitro and in vivo studies. Table
5214 summarizing the evidence regarding the antimicrobial activities of Symphytum plants in vitro.
522The reported studies present the antimicrobial activities by measuring zone diameters of growth
523inhibition. Tested extracts were obtained by using different solvent and several parts of plant.
524Overall, results indicated that extracts of Symphytum plants have potential antimicrobial effects
525on several bacterial strains tested, especially S. aureus as well as antifungal activity against
526Bipolaris oryzae (Knaak, Dias da Silva, Finger Andreis, & Mariana Fiuza, 2013). The results by
527Rocha et al suggested a possible biological control of the endophytic strains from S. officinale
528leaves against S. sclerotiorum (Rocha et al., 2009). In addition, Karavaev et al found that extracts
529from the comfrey leaves diminished the susceptibility to infection from Erysiphe graminis
530conidia and Puccinia graminis uredospores in wheat steam (Karavaev et al., 2001a). It should be

24531highlighted that the activity of the investigated extracts originates from a mix of compounds,
532including phenolic molecules, like caffeic and chlorogenic acids, allantoin and luteolin
533glycoside, probably due to synergic action of these compounds, as well as other biomolecules
534isolated from the plant material.
535In the past, Dolganiuc et al. demonstrated an alternative modality to antimicrobial
536inhibition by an aqueous extract obtained from roots of S. officinale on mouse peritoneal
537macrophages through the activation of respiratory burst of the cells and inhibition by synthesis of
538catalase, SOD, among others (Dolganiuc, Radu, & Olinescu, 1997).
539As aforementioned above and considering the inhibition or halt in the development of
540certain pathogen species by secondary metabolites present in the extracts of certain plants, the
541reviewed in vitro and in vivo studies regarding antimicrobial activities of Symphytum genus
542plants allow to summarize the following key points: i) investigations on a wide range of bacteria
543and fungi to assess the spectrum of such plant parts extracts are needed; ii) single or pooled parts
544of the plants are to be assessed for their antibacterial activity at increasing doses and using
545different aqueous/organic solvent extracts; iii) isolation and chemical structure determination of
546active compounds could improve the treatment of infections caused by pathogenic multidrug
547resistant bacteria; iv) the in vivo antimicrobial effect of Symphytum plants alone should be
548demonstrated.
549
5505.3 Antioxidant activities of Symphytum plants
551There is evidence regarding to the contribution of variety of reactive oxygen species
552(ROS) in the etiology and pathophysiology of human diseases, including inflammation,

25553autoimmune deceases, neurodegenerative disorders, viral infections, digestive system and etc.
554(Repetto & Llesuy, 2002; J. Sharifi-Rad, M. Sharifi-Rad, et al., 2018). Researchers have studied
555some scavengers that react with ROS directly or indirectly and defuse their reactivity to bounds
556oxidative damage (Barthomeuf, Debiton, Barbakadze, & Kemertelidze, 2001). Potential of
557natural compounds as antioxidant agents are topic of current interest that is being studied by
558antioxidant properties in vitro and in biological systems (Repetto & Llesuy, 2002). Several
559natural antioxidants as active oxygen scavengers, free radical inhibitors, or reducing agents from
560different plant sources have been identified (Oktay, Yildirim, Bilaloglu, & Gülçin, 2007).
561Products comprising comfrey extracts have been used in folk medicine for different
562pharmacological problem. As they promote the immunological status, wound-healing,
563antioxidant effect and anti-inflammatory, that oxidation mechanisms and free radicals can play a
564role in these process (Mulkijanyan et al., 2009). There are several literatures that have reported
565antioxidant activity of various Symphytum species. Different plant parts including root, stem and
566leaves have investigated for antioxidant efficacy and some are listed in Table 5. Most of studies
567have been done on S. officinale root and its components, comprising rosmarinic acid as a natural
568polyphenol antioxidant; allantoin as an anti-inflammatory and autoimmune enhancer agent, and
569tannins as anti-inflammatory agents (D. B. Smith & Jacobson, 2011; Trifan et al., 2018), and
570correlated with oxidative stress (E. Neagu et al., 2011 ). Indeed, different studies demonstrated
571the noteworthy antioxidant activities of Symphytum extracts compared to known antioxidants
572(Elena Neagu, Roman, & Radu, 2010).
573It has proven that polar extracts of S. officinale have higher amounts of polyphenols, and
574demonstrated higher antioxidant effects. Comparison of comfrey root ethanolic and aqueous
575extracts has demonstrated higher DPPH and SO radical scavenging activity of ethanolic extract

26576(as strong as ascorbic acid) which possessed more phenolic content than the aqueous extract
577(Alkan et al., 2014). Similar results obtained by Sowa et al in which significant antioxidant
578activity of ethanol/water extract of comfrey root was observed, associated with high total
579phenolic content and high phenolic acids concentration, including rosmarinic, p-hydroxybenzoic,
580caffeic, chlorogenic and p-coumaric acids, especially rosmarinic acid. While water extract had a
581fewer phenolic acids and less antioxidant activity (Ireneusz Sowa et al., 2018). The same
582composition of phenolic acids have already been reported for concentrated root extract that
583showed increasing of active principle by micro and ultrafiltration of ethanolic extract, rose the
584DPPH inhibitory effect even higher than Trolox inhibition activity (Paun, Neagu, Litescu,
585Rotinberg, & Radu, 2012). The association between the content of polyphenol and flavone and
586the radical scavenging activity also has been confirmed by Neagu et al via ABTS and DPPH
587methods. As concentrated extracts of S. officinale root were obtained through ultrafiltration
588contained more biologic active principles and showed high antioxidant activity (above 90%
589DPPH inhibition) (Elena Neagu, Roman, et al., 2010). Comfrey root is a valuable source of
590phenolic compounds that provide antioxidant activity. Rosmarinic and salvianolic acids are the
591main phenolic compounds present in the extract, having the ability of scavenging free radicals
592and chelating metal ions as efficient antioxidants (Trifan et al., 2018). Polar extracts of S.
593officinale leaves also have revealed antioxidant potential. Study on methanol/water, ethyl acetate
594and water extract proved methanol extract exhibited the highest polyphenols concentration and
595most power radical scavenging activity by ABTS and DPPH methods (Nossa González, Pérez,
596Verónica, Núñez, & Elías, 2016). Antioxidant evaluation of the leaves extract in another study
597showed DPPH and superoxide radical scavenging activity of ethanolic extract is more than
598aqueous extract, was correlated with more total phenolics content (Alkan et al., 2014). There are

27599some other researches that show antioxidant activity of Symphytum plants associated with
600phenolic polymers. Recently, naturally-occurring polysaccharides have attracted great attention
601by researchers caused by their antioxidant and scavenger activities (C. Liu, Chang, Zhang,
602Zhang, & Li, 2012). Several studies on scavenger activities of purified plants polysaccharides,
603crude polysaccharide extracts from different medicinal plants proved their noteworthy
604antioxidant activities as free radicals scavenger, antioxidant enzymes enhancer, lipid oxidation
605inhibitor, and advocated that plant carbohydrates, water-soluble polysaccharide complex
606fractions can be probed as novel antioxidants (C. Liu et al., 2012; Pielesz, 2012). Their
607antioxidant activity is depended to the chemical and monosaccharide composition and molecular
608weight, as mentioned items affected their electron-donating ability. Effect of different extraction
609techniques on the characteristics, yield and antioxidative potential of polysaccharides (Ps) from
610S. officinale aerial parts have been reported by Duan et al. Maximum extraction yield, smallest
611molecular weight, upper uronic acid content and best antioxidant capacity were found in Ps
612extracted by ultrasonic-assisted technique versus hot water extraction Ps that showed lowest
613yield and antioxidant activity (Duan, Shang, Chen, Li, & Wu, 2018).
614Strong inhibition of DPPH radical (IC 50 = 0.72 µg/mL instead of 1.33, 12.20, and 3.78 for
615quercetin, BHT, and ascorbic acid, respectively) and non-enzymatic lipid peroxidation of bovine
616brain extracts (IC 50 ≈ 10 ng) has reported by a water-soluble hydroxycinnamate-derived polymer
617from S. asperum. It reduced superoxide anion generation in either the reaction of phenazine
618methosulphate with NADH and molecular oxygen (IC50 ≈ 13.4 µg/mL) or in rat PMA-activated
619leukocytes (IC 50 ≈ 5 µg/mL) powerfully. Anti-lipoperoxidative evaluation showed dose-
620dependent effect and 10 ng of the S. asperum polymer inhibited 50% of lipid damage, while the

28621IC 50 of quercetin as a reference material is 2 µg/mL. It can concluded this polymer has anti-
622peroxidant activity about 200 times higher than of the standard (Barthomeuf et al., 2001).
623Two high-molecular-weight water-soluble biopolymers, the major component was
624poly[3-(3,4-dihydroxyphenyl) glyceric acid] or poly[oxy-1-carboxy-2-(3,4-
625dihydroxyphenyl)ethylene] isolated from S. officinale roots, exhibited antioxidant activity as
626expressed in terms of reducing active oxygen species (AOS) by interfering in their formation
627procedure by polymorphonuclear neutrophils (PMN) and AOS binding directly without
628cytotoxicity effect in PMNs (V Barbakadze, Van Den Berg, Beukelman, Kemmink, & van
629Ufford, 2009). The effect of phenolic polymer fractions from S. Asperum and S. caucasicum
630roots and stems (V. V. Barbakadze et al., 2007) and leaves(VV Barbakadze et al., 2011) were
631studied by luminal and lucigenin induced chemiluminescence (CLlum and CLluc) test to
632evaluate their inhibition ability on ROS produced by human polymorphonuclear neutrophils
633(PMNs) (activated by OPZ and PMA) and also on CLluc in the cell-free hypoxanthine–xanthine
634oxidase (HX/XO) system to assay superoxide anions scavenging ability. The results exhibited a
635noticeable antioxidant activity for all fractions in which the major component was poly[3-3,4-
636dihydroxyphenyl) glyceric acid] in roots and stems extracts (V. V. Barbakadze et al., 2007) that
637already has been reported from the root of both Symphytum species (V. V. Barbakadze,
638Kemertelidze, Targamadze, Shashkov, & Usov, 2002), although this polymer was not identified
639in leaves extract (VV Barbakadze et al., 2011).
640In summary, Symphytum plants have significant antioxidant effects, due to their bioactive
641compounds, and therefore, they can be considered as a factor affecting the healing process. In
642addition, their pharmacologic effects, like anti-inflammatory and wound healing can be justified
643by its antioxidant activity mechanisms.

296445.4 Hepatoprotective effect
645Symphitum officinale (L.) Gaertn ("black root" or in Italian "radice near") is a popular
646medicine in Moldova, commonly used for chronic hepatitis and hepatocirrhosis. The alcoholic
647extract of its roots was tested in vivo on 42 albino rats (180-240g) at an oral dose of 0.4 g/kg per
648daily during 2 weeks (Matcovschi, Calistru, Cojocaru, & Vaˆlcu, 1995). The chronic toxic
649hepatitis was induced by injection of 20 ml/kg tetrachloromethane twice a week for 2 months.
650The results were compared to that of the standard drug Silibor 30 mg/kg daily for 2 weeks. The
651obtained results showed that S. officinale extract produces effects statistically significant, equal
652or superior to Silibor. The hepatoprotective effects are: the decreasing of lipodystrophy and the
653absence of conjunctive proliferation in the interlobular grooves.
6545.5 Effect on musculoskeletal disorders
655Oberbaum et al. induced bone fractures in guinea pigs and had shown that S. officinale
656can accelerate and improve healing of fractured bones. The ulnae were fractured in of 35 guinea
657pigs under general anesthesia, the fracture site was then covered by the retracted muscles, the
658incision was sutured, and the wound was sprayed with Nobecutane. The X-ray and histology
659results showed that the animals which received either of the two dilutions of S. officinale and
660irrespective of the mode of administration, had completely mineralized new bone through the
661entire fracture site in 33% of the fractures (Oberbaum, Yakovlev, Kaufman, & Shoshan, 1994).
662Araujo et al. reported that emulsion containing 8% extract of comfrey leaves induced the repair
663of damaged tissue, increased the collagen deposition and reduced cellular inflammatory infiltrate
664by 46% in open wound in rat model (Araujo et al., 2012).

30665
6665.6 Wound healing activity
667Alkan et al. showed that S. officinale extracts have proliferative activity on 3T3 Swiss albino
668mouse fibroblast cells using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium
669bromide) and neutral red uptake (NRU) assays which may be responsible and favorable for
670wound healing (Alkan et al., 2014). The Caucasian species of comfrey – Symphytum asperum
671and S. caucasicum have been mainly used in folk medicine in the treatment of fractures and
672wounds as they contain allantoin. Allantoin was also reported to promote cell division and
673growth of the connective tissue, bones and cartilages and thus it is responsible for the wounds
674healing (Kommission, 1990). The extracts also contain hepatotoxic pyrrolizidine alkaloids which
675strongly restrict internal use of comfrey extracts. Barbakadze, et al. prepared allantoin and toxic
676pyrrolizidine alkaloids-free composition containing crude polysaccharides and novel biopolymer
677from S. asperum roots – poly[3-(3,4-dihydroxyphenyl) glyceric acid] (PDGA)and evaluated its
678pharmacological properties in vitro (anticomplementary and antioxidant assays) and in vivo
679experiments (mouse excisional wound and skin burn models). PDGA exhibited marked
680antioxidant and anticomplementary activity in contrast with polysaccharides, which displayed no
681detectable anticomplementary and antioxidant efficacy. Besides, ointment, containing 2.5%
682crude polysaccharides and PDGA was found to have significant wound healing properties, by
683efficacy not yielding to 2.5% allantoin ointment. The obtained results allow assuming with high
684degree of reliability that wound healing activity of comfrey preparations could be associated not
685only with allantoin but also with PDGA (V. Barbakadze et al., 2009). Savić et al. investigated the
686biological activity of pure allantoin and aqueous extract of the comfrey roots standardized to the
687allantoin content. Pure allantoin showed mild inhibitory effect on proliferation of epithelial

31688(MDCK) and fibroblastic (L929) cell lines using MTT test at concentrations 40 and 100 μg/ml,
689but more pronounced on MDCK cells. The aqueous extract of the comfrey root in concentrations
690higher than 40 μg/ml was significantly stimulatory for proliferation of L929 but inhibitory for
691MDCK cells. Pharmaceutical preparations that contained the aqueous extract showed better anti-
692irritant potential than pure allantoin by measuring electrical capacitance, erythema index (EI) and
693trans-epidermal water loss of artificially irritated skin of young healthy volunteers, 3 and 7 days
694after application. Creams showed better effect on hydration and EI compared with the gels that
695contained the same components. These results indicated that the biological activity of comfrey
696root extract is not only attributed to allantoin but is based on the different compounds present in
697the aqueous extract. Topical preparations that contain comfrey extract may have a great
698application in the treatment of skin irritation (Vesna Lj Savić et al., 2015).
699
7005.7 Anti-inflammatory and antinociceptive activity
701Vostinaru et al. evaluated the anti-inflammatory and antinociceptive activity of a S.
702officinale root extract, which was HPLC-standardized to contain 74.77 μg/mL rosmarinic acid.
703The anti-inflammatory effect of the hydro-glyceroalcoholic extract (500 mg/kg, orally) was
704assessed in the carrageenan-induced rat paw edema where the extract caused 55.6% reduction in
705edema at 1h after inflammation was induced. The antinociceptive effect, the extract caused
70645.25% inhibition of the abdominal constrictions induced by acetic acid in mice and in the
707Randall Selitto test in rats, the oral administration of the same dose demonstrated a strong
708peripheral antinociceptive effect in the first two hours after administration increasing the pain
709threshold by 58% (Vostinaru et al., 2017). A glycopeptide isolated from the aqueous extract of S.
710officinale roots exerted a dose-dependent antichloristic effect on carrageenan-induced rat paw

32711edema, inhibited the release of prostaglandins and leukotrienes via a decreased expression of
712phospholipase A2 (Hiermann & Writzel, 1998). It was reported that the anti-inflammatory
713activity of S. officinale is due to its triterpenes content, which inhibit different stages of
714inflammatory reaction including histamine release, COX and LOX activity, NO production and
715also to high polyphenol content due to selective inhibition of COX-2 (Grigore, Pirvu, Bubueanu,
716Minerva, & Rasit, 2015).
717
7186. Clinical effectiveness of Symphytum plants in human
719A monograph of the European Scientific Cooperative on Phytotherapy Monograph
720(ESCOP) stated that comfrey root was used for tendinitis, knee joint injuries, gonarthrosis,
721fractures and skin inflammation (ESCOP 2009). Preparations of the Symphytum species (most
722commonly S. officinale and S. x uplandicum) have strong clinical records which substantiate
723their longstanding traditional topical use in the treatment of musculoskeletal problems such as
724osteoarthritis (OA), back pain, ankle sprains, joint distortion, myalgia, and rheumatism. Clinical
725effectiveness of the topical preparations containing either pure extract from the underground
726parts of the comfrey plants (known as Symphyti radix) or their combinations with marketed
727formulas have been well established through individual case reports, clinical trials and post-
728marketing surveillance (Table 6).
729
7306.1. Wound healing
731Chronic marginal parodontopathies

33732A clinical and microbiological study conducted by Gafar et al reported the antimicrobial effects,
733as well as antimycotic, anti-inflammatory effects of a complex original products based on
734propolis and Symphytum off. extracts in the treatment of in the treatment of chronic marginal
735parodontopathies (Gafar, Dumitriu, Dumitriu, & Guti, 1989).
736
737Blunt injuries
738Blunt injuries are also one of the most frequent areas of application for topical comfrey
739preparations due to anti-inflammatory and analgesic effects as well as activity in stimulating
740granulation and tissue regeneration. The percutaneous efficacy of the aforementioned
741commercial product (Kytta-Salbe® f) containing 35% liquid comfrey root extract was evaluated
742in a double-blind, multicenter, randomized, placebo-controlled study on 142 patients suffering
743from unilateral acute ankle sprains (Koll et al., 2004). Patients received four treatments per day
744for 8 days. Compared to placebo, the comfrey treatment was clinically and significantly superior
745regarding the reduction of pain and ankle edema, ankle mobility and global efficacy. Under
746active treatment, no drug-related side effects were reported. In general, the trial confirmed the
747pronounced analgesic, anti-inflammatory, and anti-exudative properties as well as the good
748tolerance of the comfrey product.
749
750Acute blunt traumata
751Fresh abrasions: A study design (verum vs. reference) was used to assess wound healing effects
752of the preparation (Traumaplant®) in a randomized, double-blind clinical study including 278

34753patients with fresh abrasions (Milos Barna, Kucera, Hladícova, & Kucera, 2007). A highly
754significant and clinically relevant faster initial reduction of wound size (49 ± 19 % versus 29 ±
75513 % per day) was obtained in the favor of verum after just 2–3 days of application of the
756medication. By the physicians, the efficacy was rated from good to very good in 93.4 % of cases
757in verum group as compared to 61.7 % of cases in the group treated with the reference product.
758In case of drug related adverse reactions, none of the patients experienced problems for 10 days
759observation period. Barna et al further confirmed the wound healing effects of the topically
760applied preparation Traumaplant® containing a 10% active ingredient from the aerial parts of
761medicinal comfrey (Symphytum × uplandicum Nyman) (M Barna, Kucera, Hladikova, & Kucera,
7622012). Data for evaluating the efficacy and tolerability of the medicinal comfrey preparation
763(Traumaplant®) from the aerial parts of the plant have been also available in children. In an open
764observational study, the therapeutic applicability and safety of the product were examined in 196
765children aged from 4 to 12 years for treatment of acute blunt traumata (contusions, strains and
766distortions). Clear improvement in the range of 84.5 to 100% was found for every individual
767parameter such as pain on palpitation, pain in motion, functional impairment, edema and
768hematoma. At the same time an excellent tolerability and compliance were observed (Grünwald,
769Bitterlich, Nauert, & Schmidt, 2010). Later, the results of this non-interventional study was
770confirmed and extended with a randomized, controlled, double-blind clinical study (M Barna et
771al., 2012). The study population consisted of 108 children aged 3–12 years (n = 54/group) with
772fresh abrasions. The design of the study included the comparison of the 10% extract
773(Traumaplant®) with a low dosed 1% reference. A 50 % healing rate was reached 0.9 days
774earlier in higher concentration treatment group as compared to lower concentration group.
775Physicians and children/parents rated the efficacy of the 10% cream as significantly better than

35776that of the control preparation (1% formulation). No drug-related adverse effects or problems
777with tolerability such as local skin irritations were reported. The results of both observational and
778clinical studies in pediatric patients mentioned above demonstrated the effective and safe use of
779topical comfrey extract from the aerial parts of Symphytum for the treatment of blunt traumata.
780However, in view of safety concerns controlled/multi-center clinical trials are needed on the use
781of comfrey extracts in children.
782
783Venous ulcers
784More recently In the treatment of venous ulcers of elderly, Oreščanin reported an achievement
785within two months of the topical treatment with mix herbal wound healing ointment (Bioapifit®)
786resulted in strong wound healing, hemostatic, anti-inflammatory and antimicrobial potential
787(Visnja. Orescanin, 2016). Same author suggests the alternative use to the topical corticosteroids
788and immunomodulant therapy of mixed herbal ointment in the treatment of mild to severe atopic
789dermatitis in infants and children. Two months of the topical treatment with ointment containing
790Avena sativa, Nigella sativa, Argania spinosa, Prunus amygdalus, Daucus carota, Helichrysum
791italicum, Calendula officinalis, Matricaria chamomilla, Bellis perennis, Lavandula officinalis,
792Achillea millefolium, Thymus serpyllum, Salvia officinalis, S. officinale, Plantago major, Olea
793europaea, Melaleuca alternifolia, Cera alba, honey, and glycerol resulted in complete and/or
794strict remission of symptomatology (Visnja. Orescanin, 2016). Oreščanin attributed the effect to
795the ointment’s formulation containing emollients, strong anti-inflammatory, immunomodulating,
796wound healing and antimicrobial agents of herbal origin that simultaneously targeted the
797multiple mechanisms involved in atopic dermatitis pathogenesis. In a randomized double-blind

36798trial conducted in children (Visnja. Orescanin, 2016). In a pilot study, Binic´ et al evaluated the
799healing and antimicrobial effects of herbal therapy on venous leg ulcer. The topical herbal
800treatment (Plantoderm® ointment) containing alcohol extracts of Calendulae off., Symphytum
801off., Achilea millefolium and Salvia off. counteracted polymicrobiological effects involving S
802aureus, P. aeruginosa, E coli and P mirabilis on non-infected venous leg ulcer (Binic I, A, D, I,
803& Z., 2010).
804
805Squamous endocervical metaplasia
806Oreščanin et al tested the efficiency of newly developed herbal pessaries containing macerates
807(Calendula officinalis, Matricaria chamomilla, Lavandula officinalis, Hypericum perforatum L.,
808Achilea millefolium, Thymus serpyllum, Salvia officinalis, Mentha piperita L., S. officinale,
809Plantago major L., Alchemilla vulgaris) and essential oils (Melaleuca alternifolia, Thymus
810vulgaris, Pelargonium graveolens, Cympobogon martinii, and Origanum vulgare L.) for the
811treatment of the squamous endocervical metaplasia in women aged 20-59 years (Orescanin,
812Guštek Štefica, & Krivak Bolanca, 2015). The wound healing, anti-inflammatory, antimicrobial
813and antiviral effects of the pessaries could be due to the synergistic effect of pooled medicinal
814plants. In a more recent conference paper (2017) the application of herbal ointment
815(BIOAPIGYN ®) for the treatment of lower genital tract infections was successful against U.
816urealyticum and E. coli while no effects versus M. hominis, and Candida. The achievement goal
817probably due to the significant prebiotic and probiotic activity and low pH of the ointment
818constituents including oil extracts of Achillea millefolium, Thymus serpyllum, Salvia officinalis,
819Mentha piperita, Alchemilla vulgaris, Olea europaea; essential oils: Melaleuca alternifolia,

37820Thymus vulgaris ct. thymol, Cinnamomum camphora ct. cineol, Cympobogon martini, Origanum
821compactum and Eugenia caryophyllata; Avena sativa, Cera flava; honney; and
822glycerolmedicinal (Orescanin et al., 2015).
823
824Skin disorders
825Comfrey extracts have also been used in the treatment of some skin diseases such as erythema,
826chronic varicose ulceration and decubital ulcers. Although there is considerable amount of
827clinical and observational data in the treatment of musculoskeletal diseases, the use of comfrey
828extracts in skin disorders is limited with experimental studies and individual case reports. In a 29
829volunteers, the effects of topical preparations containing 5 or 10 % of a comfrey root extract (2:7,
83050 % ethanol) were studied on the process of healing of experimentally induced UV-B erythema
831(Andreas, Brenneisen, & Clerc, 1989). The anti-inflammatory potency of the extract was found
832to be equal to or greater than that of diclofenac. Recently, the efficacy of 10% topical comfrey
833extract preparation (Traumaplant®) was evaluated in an open, prospective study with the
834involvement of 161 patients who were in the second and third stage of decubitus ulcers (pressure
835ulcers, ITT population). The bandages with the cream were used for application and were
836changed for every 2–3 days for 4 weeks. The primary efficacy parameters were the area of the
837sore and the depth of the wound (planimetrically in mm). Accordingly, in case of total decubitus
838area 89.2% reduction was observed and similar result (88% reduction) was also obtained for the
839depth of the pressure ulcer. Only two cases of local irritation were observed after treatment
840period. Therefore, this study confirms that Symphytum herb extract is highly effective in the
841treatment of pressure ulcers and also it shows excellent skin compatibility in case of open sores

38842(Štepán, Ehrlichova, & Hladikova, 2014). However, taking into consideration the general rule,
843randomized, controlled clinical trials are crucial to justify the efficacy and safety of comfrey
844extracts in the treatment of skin problems.
845
8466.2. Musculoskeletal problems
847Osteoarthritis
848Randomized controlled clinical trials were performed on the use of different comfrey root
849preparations in the management of osteoarthritis (OA) related symptoms. According to World
850Health Organization, half of the world population aged 65 years and older has OA, which is the
851most prevalent disorder of articulating joints in human and is restricting quality of life.
852Therefore, reduction of pain, preservation and the restoration of the joint’s function are important
853therapeutic objectives (Bhatia, Bejarano, & Novo, 2013). Smith and Jacobson studied the effect
854of 10% or 20% comfrey root extract-based cream containing a blend of tannic acid and
855eucalyptus oil compared to a placebo cream and a standard cream containing eucalyptus only on
856forty-three male and female subjects (45-83 years old) with diagnosed primary osteoarthritis of
857the knee. Participants used the cream 3 times daily for 6 weeks and were evaluated every 2
858weeks during the treatment. The results showed significant differences in osteoarthritis index
859categories at p˂0.01 (pain, stiffness and physical functioning), confirming that the two
860concentrations of comfrey-based creams were superior to the Eucalyptus reference cream. Two
861participants in each group had temporary and minor adverse reactions of skin rash and itching,
862which were rapidly resolved by modifying applications (Smith and Jacobson 2011). The earliest

39863randomized, placebo-controlled, clinical trial was carried out on 61 patients with OA knee by
864using a cream formulation containing comfrey root extract (unknown species) and mistletoe. 1.7
865g verum or placebo (identical cream without comfrey and mistletoe extracts) was applied twice a
866day to patients. Significantly greater reduction was observed in morning/evening pain (both
86728%) and night pain (51%) in herbal group as compared to placebo (Schmidtke-Schrezenmeier,
8681992). A double-blind, randomized, placebo-controlled, bi-center clinical trial was conducted
869over a period of 21 days with 220 painful knee OA patients (Grube, Grünwald, Krug, & Staiger,
8702007). All patients received either a commercial preparation (Kytta-Salbe® f; Merck
871Selbstmedikation GmbH, Darmstadt, Germany) containing comfrey root extract (1:2, ethanol
87260%, V/V, 35%) or a corresponding placebo cream three times a day. At the end of the trial, pain
873in the verum group had reduced five times (reduced from moderate to mild) more than in the
874placebo group. The primary target variable was the VAS (Visual analogue scale: total score of
875pain at rest and pain on movement) improved 54.7% in the verum group and only 10.7% in the
876placebo group. Regarding secondary target value-WOMAC total score (Western Ontario and
877McMaster Universities Osteoarthritis Index) which measures pain, stiffness and functional
878limitation, verum proved to be four times more effective than the placebo. Overall, the trial was
879found to be well conducted in accordance with the GCP-ICH guidelines and in case of its
880findings the short-term usage of the product could be an effective treatment strategy for painful
881OA knee. Another randomized controlled, multicenter clinical trial conducted on 43 patients with
882OA knee included the application of a marketed comfrey-based botanical cream (4Jointz, PO
883Box 5218, Brisbane 4001, Australia) containing a blend of tannic acid and eucalyptus, three
884times a day for 6 weeks (D. B. Smith & Jacobson, 2011). The two concentrations of the comfrey
885root extract (10% and 20% strength by volume) ointments were compared with a pseudo-placebo

40886of eucalyptus similar in color, texture, and smell to the experimental ointments. Results were
887evaluated every 2 weeks during the treatment. 10% and 20% formula were both effective in
888relieving pain and stiffness and in improving daily function within the first 2 weeks of treatment.
889This improvement continued at each 2-week assessment period for the duration of the study. For
8903 variables addressed, both 10% and 20% comfrey-based creams were superior to the eucalyptus
891reference cream. Two participants had temporary and minor adverse reactions of skin rash and
892itching, which were rapidly resolved by modifying applications. In a bi-center, randomized,
893double-blind, parallel group, placebo-controlled trial a different combination of 4Jointz cream
894containing a standardized comfrey root extract (200 mg/g) and pharmaceutical grade tannic acid
895(100 mg/g) plus aloe vera gel (300 mg/g), eucalyptus oil (40 mg/g), and frankincense oil (1.0
896mg/g) were tested on 133 OA patients (Laslett et al., 2012). Several parameters including pain,
897markers of inflammation and cartilage breakdown had been evaluated over 12 weeks. At the end
898of the treatment, pain scores significantly decreased in the group who received 4Jointz compared
899to group who received placebo. Scores were assessed by using both the VAS pain intensity and
900the KOOS (Knee Injury and Osteoarthritis Outcome Score) pain scale. Changes in IL-6 and
901CTX-2 were not significant. Therefore, it can be inferred that topical treatment using 4Jointz
902reduced pain but had no effect on inflammation or cartilage breakdown in 12 weeks of treatment.
903Overall, the findings of all these randomized, controlled clinical trials support the efficient use of
904comfrey root extract in the symptomatic treatment of OA and also establish its place as an
905alternative option to other topical preparations (Laslett et al., 2012).
906
907Back pain

41908Formulations containing comfrey root extracts have been also effective alternative for the
909treatment of back pain, a very common health problem affecting physical performance and
910general well-being worldwide. A double-blind, multicentre, randomized clinical trial with
911parallel group design was conducted on 120 patients with acute upper and lower back pain over a
912period of 5 days (Giannetti, Staiger, Bulitta, & Predel, 2009). The patients were administered
913with 4 g of licensed medicinal product (Kytta-Salbe® f-mentioned above) containing comfrey
914fluid root extract or placebo per application, three times a day. The trial included four visits.
915According to results, there was a significant treatment difference between comfrey root extract
916and placebo regarding the primary efficacy variable which was defined as the area under the
917curve (AUC) of the VAS on active standardized movement values at visits 1 to 4. During the
918trial, the intensity of the pain on active standardized movement decreased approximately 95.2%
919in the verum group and 37.8% in the placebo group. Also, with this study a fast-acting effect of
920the comfrey root extract has been addressed for the first time. In comfrey group the pain intensity
921was reduced about 33% after 1h, however, the rate of the decrease was just 12% in placebo
922group. Comfrey root extract showed a remarkably potent fast acting effect which was free of
923serious adverse reactions and was clinically relevant in reducing acute back pain. The
924combination of the same comfrey root extract cream with 1.2% methyl nicotinate (Kytta-Balsam
925Wf, Merck Selbstmedikation GmbH, Darmstadt, Germany) was investigated in a randomized,
926multicentre, double-blind, three-arm, placebo-controlled trial (Pabst, Schaefer, Staiger,
927Junker‐Samek, & Predel, 2013). In this study, the combination product was compared with a
928single preparation of methyl nicotinate or placebo cream for relief of acute upper or low back
929pain in 379 patients who are administered by a cream three times daily over 5 days. The results
930demonstrated that patients treated with the topical combination of comfrey root extract and

42931methyl nicotinate had statistically significant and clinically relevant reductions in pain scores and
932increases in tenderness. As a result, the clinical trial presented that methyl nicotinate contributed
933substantially to the efficacy of the combination product, reduced the primary efficacy parameter
934by 31% compared to placebo. The 22 drug-related adverse effects, which were classified as mild
935to moderate, occurred in combination and methyl nicotinate groups; however, none in placebo
936group. Pancreatic insufficiency and recurrent depression were two series adverse effects
937observed in combination treatment group.
938
939Epicondylitis, tendovaginitis, and periarthritis
940In one of the earliest randomized pilot study, 41 patients with different forms of musculoskeletal
941rheumatism (mainly epicondylitis, tendovaginitis, and periarthritis) were administered with a
942comfrey root ointment (n=20) or placebo (n=21) for four weeks (Petersen, Lorkowski, Kasper,
943Gottwald, & Lücker, 1993). Patients were assessed by using different pain parameters such as
944tenderness on pressure, pain at rest and on exercise. At the end of 2 weeks, treatment of patients
945with epicondylitis and tendovaginitis resulted in a clearly better efficacy of the comfrey ointment
946as compared to placebo regarding "tenderness on pressure". However, treatment of patients with
947periarthritis did not show any positive effect compared with placebo even at the end of the four
948weeks.
949
950Acute ankle distortion

43951In a single-blind, controlled, randomized, parallel-group, multicenter and confirmatory clinical
952trial, patients with acute unilateral ankle sprains received either a commercial product (Kytta-
953Salbe®-f) containing 35 % liquid comfrey root extract (n=82) or Diclofenac gel (n=82)
954containing 1.16 g of diclofenac diethylamine salt four times a day, during 7 days (Predel,
955Giannetti, Koll, Bulitta, & Staiger, 2005). The aim of this study was to compare percutaneous
956efficacy and tolerability of Comfrey ointment with that of Diclofenac gel which penetrates the
957skin barrier to reach joints, muscles and synovial fluid and exerts highly potent, local therapeutic
958activity (Assandri, Canali, & Giachetti, 1993; Radermacher, Jentsch, Scholl, Lustinetz, &
959Frolich, 1991; Riess et al., 1986). In this study, the primary efficacy variable was determined as
960pain arising from pressure on the injured area and evaluated by the AUC of the pain-time curve.
961Secondary variables were defined as circumference of the joint, the individual pain sensation
962while resting and in movement. According to all results, considering both primary and secondary
963variables, Comfrey extract was found to be superior to Diclofenac gel. After 7 days treatment,
964the ankle swelling decreased 79.5% in the comfrey root extract group, however, decrease was
96569.4% in the diclofenac group. While the pain on pressure measured by algometer was reduced
96680.6% by the comfrey root extract, it decreased 74.7% in diclofenac group. By considering
967several different parameters, the re-evaluation of the trial according to CPMP- guidelines also
968exhibited superiority of the comfrey preparation over diclofenac gel (EMA, 2000). Another
969controlled, double blind, randomized multi-center clinical trial assessed the efficacy and safety of
970a special topical preparation (Traumaplant®) containing 10% comfrey extract (as active
971ingredient) from the aerial parts of Symphytum × uplandicum Nyman in 203 patients with acute
972ankle distortion (Kučera, Barna, Horáček, Kováriková, & Kučera, 2004). The efficacy and
973tolerability were compared with a reference product containing 1% active ingredient. Potentially

44974toxic PAs were not present in the preparations for both verum and reference groups (detection
975limit < 0.1 μg/g). In this study, reduction of pain on active motion, pain at rest and functional
976impairment was found to be highly significant and clinically relevant on days 3, 4 and 7 with the
977use of high concentration product (p <0.001). Amelioration of swellings as compared to
978reference was also significant on day 3–4 (p < 0.01). Accordingly, the overall efficacy was
979determined good to excellent in 85.6 % of cases with verum compared to 65.7 % of cases with
980reference on day 3–4. Tolerability was found to be excellent. Overall, the study results confirmed
981the positive benefit-risk ratio and underlined that a sufficiently high extract concentration is
982needed for an optimal effect. In one such open uncontrolled study, the effects of comfrey extract
983obtained from the aerial parts of the plant were investigated. The ointment was applied to 105
984patients with painful disorders of the locomotor system twice a day. Very effective results were
985obtained in the treatment of subacute and chronic complaints accompanied by functional
986symptoms like swellings (excellent results in 90–94 % of patients). Even in a subset of patients
987(57 out of 105) functional disturbances and pain were completely resolved (Kucera et al., 2000).
988
989Myalgia
990The effectiveness and tolerability of the same topical product (Traumaplant®-mentioned above
991with 10% active ingredient) were tested in a double-blind, reference-controlled, randomized,
992multicenter study containing 215 patients with myalgia (Kucera et al., 2005). Results were
993compared with a reference product containing 1% active ingredient. The primary efficacy
994parameter was pain in motion assessed with the aid of a visual analogue scale. The secondary
995efficacy parameters included pain at rest, pain on palpation, and functional impairment. With

45996high concentrations of the treatment product, pain on active motion, pain at rest and pain on
997palpation were significantly better reduced than with the reference product. It was clinically
998relevant that the higher concentration of Symphytum active ingredients in the study medication
999was responsible for a quicker decrease in pain as compared with low-dose Symphytum.
1000Tolerability of the study medication was assessed as excellent since no systemic adverse effects
1001were observed.
1002The results of all these randomized, controlled clinical trials established the efficient use
1003of comfrey root extract in the treatment of musculoskeletal problems and provided evidences for
1004being a more potent option compared with other topical treatments. Despite the effectiveness of
1005comfrey root extract, there are some concerns related with its safety. Comfrey roots contain 0.2-
10060.4% pyrrolizidine alkaloids (PAs): symphytine, lycopsamine/intermedine (diastereoisomers),
1007acetyl-lycopsamine/acetyl-intermedine (diastereoisomers), myoscorpine, lasiocarpine,
1008heliosupine, viridiflorine, echiumine, symlandine and echimidine (Barnes, Anderson, &
1009Phillipson, 2007; Coulombe Jr, 2003). PAs may also be present in the comfrey plants as N-
1010oxides forms. Both PAs and their N-oxides are biologically inactive compounds without any
1011toxicity. However, in body they are metabolically activated by hepatic cytochrome P450 (CYPs)
1012drug metabolizing enzymes, specifically CYP3A and CYP2B isoforms (Stickel & Seitz, 2000).
1013These reactions yield corresponding pyrrolic metabolites that can react with cellular
1014macromolecules, such as proteins and DNA leading to genotoxic and/or carcinogenic effects (A.
1015S. Prakash, Pereira, Reilly, & Seawright, 1999). Although internal use of comfrey root extracts is
1016traditionally recommended for the treatment of rheumatoid arthritis, bronchitis, various allergies,
1017diarrhea, gastritis and gastroduodenal ulcers, its efficacy and safety have never been
1018demonstrated in controlled investigations. Moreover, numerous case reports have shown that

461019internal use of comfrey extracts are associated with severe hepatotoxicity particularly cirrhosis
1020and ascites (Stickel & Seitz, 2000). For this reason, the internal use of comfrey has not been
1021advised and been restricted in the USA and Canada (EMA, 2005). In most of the European
1022countries such as Germany, France and England, the use of comfrey root is just limited to
1023external products. According to the Commission E, the daily exposed dose should not exceed
1024more than 100 μg PA with 1,2 unsaturated necine structure, including its N-oxides (EMA, 2005).
1025Despite the fact that percutaneous absorption of PAs present in comfrey species is low (Barnes et
1026al., 2007), the duration of treatment should not be longer than 4-6 week per year (Blumenthal &
1027Busse, 1998) and application of comfrey preparations to the broken skin should be avoided. Most
1028of the clinical trials mentioned above were performed by using fully licensed medicinal products
1029containing comfrey root extract, which is either PA-free or -depleted. The use of these processed
1030root extracts has been proved to be safe with numerous controlled trials-the numbers of drug
1031related adverse reactions were very low and not serious. However, the use of traditional
1032preparations with comfrey root extract may represent a potential health hazard. Since the total
1033PAs concentrations in above-ground plant parts especially in the leaves are considerably lower
1034(15-55 μg/g leaf) than the contents of roots (1380- 8320 μg/g root), the use of aerial parts of the
1035comfrey can be a safer alternative for self-medication (Carole E Couet, Colin Crews, & A Bryan
1036Hanley, 1996).
1037Overall, as previously report by Frost et al. in a critical scoping review (Frost et al., 2013)
1038randomized controlled trials, non-randomized controlled trials and observational studies showed
1039evidence of benefit of comfrey for ankle distortion, back pain, abrasion wounds and
1040osteoarthritis with few adverse events. Topical application proved safety, but further rigorous
1041assessment is needed the clinical trials and observational data at hand substantiate the traditional

471042topical use of Symphytum species in the treatment of musculo-skeletal problems and blunt
1043injuries. No or very minor adverse effects were reported in these studies. However, it should be
1044noted that most of the controlled clinical trials mentioned in this review were performed on fully
1045licensed PA-depleted or PA-free medicinal products distributed on the markets. Therefore,
1046patients who rely on traditional comfrey preparations should be aware of the potential health
1047hazards.
1048
10497. Conclusions and Future perspectives
1050The current reviews on Symphytum plants explore the evidence for a range of indications.
1051However most of data are descriptive overviews of clinical study findings from manufacturer
1052with little information on search strategy or risk of bias for each study. Other aspect relating to
1053Symphytum plants focus chiefly on the potential for pyrrolizidine alkaloids toxicity, though
1054numbers of biologically active polymers, saccharides, phenolic compounds and terpenoids.
1055However, the information currently available is not sufficient to allow an accurate assessment of
1056the risks or potential therapeutic benefits of comfrey. Nevertheless, ancient herbalists used them
1057in specific way with doses adequacy as well as the synergistic use more than single plants and
1058nowadays only pyrrolizidine-depleted extracts are used in topical medicinal products. It is
1059important to note that fully licensed medicinal products available today contain depleted or PA-
1060free processed extracts. Indeed, clinical trials support the traditional topical use of Symphytum
1061species in the treatment of musculoskeletal problems and blunt injuries with minor adverse
1062effects. Symphytum was poorly investigated for its antimicrobial potency, and thus, further
1063investigations are needed to assess the antimicrobial spectrum of Symphytum plants, that should

481064normally include either single or pooled plant parts at increasing doses and using different
1065aqueous/organic solvent extracts, characterizing active natural products both in vitro and in vivo.
1066
1067Acknowledgements
1068Natália Martins would like to thank the Portuguese Foundation for Science and Technology
1069(FCT–Portugal) for the Strategic project ref. UID/BIM/04293/2013 and “NORTE2020 –
1070Programa Operacional Regional do Norte” (NORTE-01-0145-FEDER-000012).
1071
1072Conflict of interests
1073The authors declare no conflict of interest.
1074
1075References
1076Abdolshahi, A., Naybandi-Atashi, S., Heydari-Majd, M., Salehi, B., Kobarfard, F., Ayatollahi, S., . . .
1077Montanari, C. (2018). Antibacterial activity of some Lamiaceae species against Staphylococcus
1078aureus in yoghurt-based drink (Doogh). Cellular and molecular biology (Noisy-le-Grand,
1079France), 64(8), 71-77.
1080Abou-Mandour, A. A., Czygan, F. C., Haaß, D., & Franz, G. (1987). Fructan synthesis in tissue cultures
1081of Symphytum officinale L.: initiation, differentiation, and metabolic activity. Planta Med, 53,
1082482-487.
1083Aceves-Avila, F. J., Medina, F., & Fraga, A. (2001). Herbal therapies in rheumatology: the persistence of
1084ancient medical practices. Clin Exp Rheumatol, 19(2), 177-183.
1085Adeneye, A. A. (2014). 6 – Subchronic and Chronic Toxicities of African Medicinal Plants. In V. Kuete
1086(Ed.), Toxicological Survey of African Medicinal Plants (pp. 99-133): Elsevier.
1087Ahmad, V. U., Noorwala, M., Mohammad, F. V., Sener, B., & Aftab, K. (1993). Symphytoxide A, a
1088triterpenoid saponin from the roots of Symphytum officinale. Phytochemistry, 32, 1003-1006.
1089Al-Nimer, M. S., & Wahbee, Z. (2017). Ultraviolet light assisted extraction of flavonoids and allantoin
1090from aqueous and alcoholic extracts of Symphytum officinale. Journal of Intercultural
1091Ethnopharmacology, 6, 280-285.
1092Alkan, F. U., Anlas, C., Ustuner, O., Bakırel, T., & Sari, A. B. (2014). Antioxidant and proliferative
1093effects of aqueous and ethanolic extracts of Symphytum officinale on 3T3 Swiss albino mouse
1094fibroblast cell line. Asian J Plant Sci Res, 4(4), 62-68.

491095Altamirano, J. C., Gratz, S. R., & Wolnik, K. A. (2005). Investigation of pyrrolizidine alkaloids and their
1096N-oxides in commercial comfrey-containing products and botanical materials by liquid
1097chromatography electrospray ionization mass spectrometry. Journal of AOAC International, 88,
1098406-412.
1099Amakura, Y., Okada, M., Tsuji, S., & Tonogai, Y. (2000). High-performance liquid chromatographic
1100determination with photodiode array detection of ellagic acid in fresh and processed fruits. J
1101Chromatogr A, 896(1-2), 87-93.
1102Andreas, P., Brenneisen, R., & Clerc, J. (1989). Relating antiphlogistic efficacy of dermatics containing
1103extracts of Symphytum officinale to chemical profiles. Planta Medica, 7, 55-66.
1104Araujo, L. U., Reis, P. G., Barbosa, L. C., Saude-Guimaraes, D. A., Grabe-Guimaraes, A., Mosqueira, V.
1105C., . . . Silva-Barcellos, N. M. (2012). In vivo wound healing effects of Symphytum officinale L.
1106leaves extract in different topical formulations. Pharmazie, 67(4), 355-360.
1107Assandri, A., Canali, S., & Giachetti, C. (1993). Local tolerability and pharmacokinetic profile of a new
1108transdermal delivery system, diclofenac hydroxyethylpyrrolidine plaster. Drugs under
1109experimental and clinical research, 19(3), 89-95.
1110Avancini, C., Wiest, J. M., Dall’Agnol, R., Haas, J. S., & von POSER, G. L. (2008). Antimicrobial
1111activity of plants used in the prevention and control of bovine mastitis in Southern Brazil. Latin
1112American Journal of Pharmacy, 27(6), 894-899.
1113Avula, B., Sagi, S., Wang, Y. H., Zweigenbaum, J., Wang, M., & Khan, I. A. (2015). Characterization
1114and screening of pyrrolizidine alkaloids and N-oxides from botanicals and dietary supplements
1115using UHPLC-high resolution mass spectrometry. Food Chemistry, 178, 136–148.
1116Badridze, G., Kacharava, N., Chkhubianishvili, E., Rapava, L., Kikvidze, M., Chigladze, L., &
1117Chanishvili, S. (2013). Content of antioxidants in leaves of some plants of Tbilisi environs. Bull.
1118Georg. Natl. Acad. Sci, 7(3).
1119Bala, I., Bhardwaj, V., Hariharan, S., & Kumar, M. N. (2006). Analytical methods for assay of ellagic
1120acid and its solubility studies. J Pharm Biomed Anal, 40(1), 206-210.
1121doi:10.1016/j.jpba.2005.07.006
1122Barbakadze, V., Kemertelidze, E., Targamadze, I., Mulkijanyan, K., Shashkov, A. S., & Usov, A. I.
1123(2005). Poly[3-(3,4-dihydroxyphenyl)glyceric acid], a new biologically active polymer from
1124Symphytum asperum Lepech. and S. caucasicum Bieb. (Boraginaceae). Molecules, 10, 1135-
11251144.
1126Barbakadze, V., Mulkidzhanyan, K., Merlani, M., Gogilashvili, L., Amiranashvili, L. S., & Shaburishvili,
1127E. (2011). Extraction, composition and the antioxidant and anticomplement activities of high
1128molecular weight fractions from the leaves of Symphytum asperum and S. caucasicum.
1129Pharmaceutical Chemistry Journal, 44(11), 604-607.
1130Barbakadze, V., Mulkijanyan, K., Gogilashvili, L., Amiranashvili, L., Merlani, M., Novikova, Z., &
1131Sulakvelidze, M. (2009). Allantoin-and Pyrrolizidine Alkaloids-Free Wound Healing
1132Compositions from Symphytum asperum. Bulletin of the Georgian National Academy of
1133Sciences, 3(1), 159-164.
1134Barbakadze, V., Mulkijanyan, K., Merlani, M., M. Gogilashvili, L., Amiranashvili, L., & K.
1135Shaburishvili, E. (2011). Extraction, composition and the antioxidant and anticomplement
1136activities of high molecular weight fractions from the leaves of Symphytum asperum and S.
1137caucasicum. Pharmaceutical Chemistry Journal, 44, 604-607. doi:10.1007/s11094-011-0527-9
1138Barbakadze, V., Van Den Berg, A., Beukelman, C., Kemmink, J., & van Ufford, H. Q. (2009). Poly [3-(3,
11394-dihydroxyphenyl) glyceric acid] from Symphytum officinale roots and its biological activity.
1140Chemistry of Natural Compounds, 45(1), 6-10.
1141Barbakadze, V. V., Kemertelidze, E. P., Mulkijanyan, K. G., Van Den Berg, A. J. J., Beukelman, C. J.,
1142Van Den Worm, E., . . . Usov, A. I. (2007). Antioxidant and anticomplement activity of poly[3-
1143(3,4-dihydroxyphenyl) glyceric acid] from Symphytum asperum and Symphytum caucasicum
1144plants. Pharmaceutical Chemistry Journal, 41, 14-16.

501145Barbakadze, V. V., Kemertelidze, E. P., Mulkijanyan, K. G., Van Den Berg, A. J. J., Beukelman, C. J.,
1146Van Den Worm, E., . . . Usov, A. I. (2007). Antioxidant and anticomplement activity of poly[3-
1147(3,4-dihydroxyphenyl) glyceric acid] from Symphytum Asperum and Symphytum Caucasicum
1148plants. Pharmaceutical Chemistry Journal, 41(1), 14-16. doi:10.1007/s11094-007-0004-7
1149Barbakadze, V. V., Kemertelidze, E. P., Shashkov, A. S., & Usov, A. I. (2000). Structure of a new
1150anticomplementary dihydroxycinnamate-derived polymer from Symphytum asperum
1151(Boraginaceae). Mendeleev Commun., 10, 148–149.
1152Barbakadze, V. V., Kemertelidze, E. P., Targamadze, I. L., Shashkov, A. S., & Usov, A. I. (2002).
1153Poly[3-(3,4-dihydroxyphenyl)glyceric acid]: A new biologically active polymer from two
1154comfrey species Symphytum asperum and S. caucasicum (Boraginaceae). Russian Journal of
1155Bioorganic Chemistry, 28(4), 326-330. doi:10.1023/A:1019552110312
1156Barna, M., Kucera, A., Hladícova, M., & Kucera, M. (2007). Der wundheilende Effekt einer Symphytum-
1157Herba-Extrakt-Creme (Symphytum× uplandicum Nyman): Ergebnisse einer randomisierten,
1158kontrollierten Doppelblindstudie. Wiener Medizinische Wochenschrift, 157(21-22), 569-574.
1159Barna, M., Kucera, A., Hladikova, M., & Kucera, M. (2012). Randomized double-blind study: Wound-
1160healing effects of a Symphytum herb extract cream (Symphytum× uplandicum Nyman) in
1161children. Arzneimittelforschung, 62(06), 285-289.
1162Barnes, J., Anderson, L. A., & Phillipson, J. D. (2007). Herbal medicines: Pharmaceutical Press.
1163Barthomeuf, C., Debiton, E., Barbakadze, V., & Kemertelidze, E. (2001). Evaluation of the dietetic and
1164therapeutic potential of a high molecular weight hydroxycinnamate-derived polymer from
1165Symphytum asperum Lepech. Regarding its antioxidant, antilipoperoxidant, antiinflammatory,
1166and cytotoxic properties. Journal of agricultural and food chemistry, 49(8), 3942-3946.
1167Becker, L. C., Bergfeld, W. F., Belsito, D. V., Klaassen, C. D., Marks, J. G., Shank, R. C., . . . Andersen,
1168F. A. (2010). Final report of the safety assessment of allantoin and its related complexes.
1169International Journal of Toxicology, 29, 84S-97S.
1170Bellardi, M. G., & Benni, A. (2005). The occurrence of alfalfa mosaic virus in Symphytum tuberosum L.
1171Journal of Plant Pathology, 87(1), 75.
1172Bhatia, D., Bejarano, T., & Novo, M. (2013). Current interventions in the management of knee
1173osteoarthritis. Journal of pharmacy & bioallied sciences, 5(1), 30.
1174Binic I, A, J., D, J., I, J., & Z., V. (2010). Evaluation of healing and antimicrobiological effects of herbal
1175therapy on venous leg ulcer: pilot study. Phytotherapy Research, 2010.
1176Blumenthal, M., & Busse, W. (1998). American Botanical Council. Integrative Medicine
1177Communications, Germany.
1178Bogert, L. J., Briggs, G. M., & Calloway, D. H. (1973). Nutrition and physical fitness. Philadelphia, PA.:
1179W.B. Saunders Co.
1180Borchardt, J. R., Wyse, D. L., Sheaffer, C. C., Kauppi, K. L., Ehlke, R. G. F. N. J., Biesboer, D. D., &
1181Bey, R. F. (2008). Antimicrobial activity of native and naturalized plants of Minnesota and
1182Wisconsin. Journal of Medicinal Plants Research, 2(5), 098-110.
1183Bouzada, M. L. M., Fabri, R. L., Nogueira, M., Konno, T. U. P., Duarte, G. G., & Scio, E. (2009).
1184Antibacterial, cytotoxic and phytochemical screening of some traditional medicinal plants in
1185Brazil. Pharmaceutical Biology, 47(1), 44-52.
1186Brauchli, J., Lüthy, J., Zweifel, U., & Schlatter, C. (1982). Pyrrolizidine alkaloids from Symphytum
1187officinale L. and their percutaneous absorption in rats. Experientia, 38, 1085-1087.
1188Brown, A. W., Stegelmeier, B. L., Colegate, S. M., Gardner, D. R., Panter, K. E., Knoppel, E. L., & Hall,
1189J. O. (2016). The comparative toxicity of a reduced, crude comfrey (Symphytum officinale)
1190alkaloid extract and the pure, comfrey-derived pyrrolizidine alkaloids, lycopsamine and
1191intermedine in chicks (Gallus gallus domesticus). J Appl Toxicol, 36(5), 716-725.
1192doi:10.1002/jat.3205
1193Cameron, M., & Chrubasik, S. (2013). Topical herbal therapies for treating osteoarthritis. Cochrane
1194Database Syst Rev(5), Cd010538. doi:10.1002/14651858.cd010538

511195Carocho, M., Morales, P., & Ferreira, I. C. F. R. (2018). Antioxidants: Reviewing the chemistry, food
1196applications, legislation and role as preservatives. Trends in Food Science & Technology, 71,
1197107-120. doi: https://doi.org/10.1016/j.tifs.2017.11.008
1198Cavero, R. Y., Akerreta, S., & Calvo, M. I. (2011). Pharmaceutical ethnobotany in Northern Navarra
1199(Iberian Peninsula). J Ethnopharmacol, 133(1), 138-146. doi:10.1016/j.jep.2010.09.019
1200Cavero, R. Y., & Calvo, M. I. (2015). Medicinal plants used for musculoskeletal disorders in Navarra and
1201their pharmacological validation. J Ethnopharmacol, 168, 255-259.
1202doi:10.1016/j.jep.2015.03.078
1203Chen, S., Shang, H., Yang, J., Li, R., & Wu, H. (2018). Effects of different extraction techniques on
1204physicochemical properties and activities of polysaccharides from comfrey (Symphytum
1205officinale L.) root. Industrial Crops and Products, 121, 18-25.
1206doi: https://doi.org/10.1016/j.indcrop.2018.04.063
1207Chittendon, F. (1956). RHS Dictionary of Plants plus Supplement: Oxford University Press.
1208Chou, M. W., & Fu, P. P. (2006). Formation of DHP-derived DNA adducts in vivo from dietary
1209supplements and Chinese herbal plant extracts containing carcinogenic pyrrolizidine alkaloids.
1210Toxicol Ind Health, 22, 321–327.
1211Colegate, S. M., Stegelmeier, B. L., & Edgar, J. A. (2012). 14 – Dietary exposure of livestock and humans
1212to hepatotoxic natural products. In J. Fink-Gremmels (Ed.), Animal Feed Contamination (pp.
1213352-382): Woodhead Publishing.
1214Couet, C. E., Crews, C., & Hanley, A. B. (1996). Analysis, separation, and bioassay of pyrrolizidine
1215alkaloids from comfrey (Symphytum officinale). Natural toxins, 4(4), 163-167.
1216Couet, C. E., Crews, C., & Hanley, A. B. (1996). Analysis, separation, and bioassay of pyrrolizidine
1217alkaloids from comfrey (Symphytum officinale). Nat Toxins., 4, 163-167.
1218Coulombe Jr, R. A. (2003). Pyrrolizidine alkaloids in foods.
1219Culvenor, C. C. J., Edgar, J. A., Frahn, J. L., & Smith, L. W. (1980 ). The alkaloids of Symphytum ×
1220uplandicum (Russian comfrey). Australian Journal of Chemistry, 33, 1105 – 1113
1221Cvetkovic, D., Stanojević, L., Kundaković, T., Zlatkovic, S., & Nikolić, G. (2015). Antioxidant and
1222antimicrobial activity of a new generation phyto-gel. . Advanced technologies, 4, 11-18.
1223de Albuquerque, U. P., Monteiro, J. M., Ramos, M. A., & de Amorim, E. L. (2007). Medicinal and magic
1224plants from a public market in northeastern Brazil. J Ethnopharmacol, 110(1), 76-91.
1225doi:10.1016/j.jep.2006.09.010
1226Dolganiuc, A., Radu, L., & Olinescu, A. (1997). The effect of products of plant and microbial origin on
1227phagocytic function and on the release of oxygen free radicals by mouse peritoneal macrophages.
1228Bacteriol Virusol Parazitol Epidemiol, 42(1-2), 65-69.
1229Dresler, S., Szymczak, G., & Wojcik, M. (2017). Comparison of some secondary metabolite content in
1230the seventeen species of the Boraginaceae family. Pharmaceutical Biology, 55, 691–695.
1231Duan, M., Shang, H., Chen, S., Li, R., & Wu, H. (2018). Physicochemical properties and activities of
1232comfrey polysaccharides extracted by different techniques. Int J Biol Macromol, 115, 876-882.
1233EFSA. (2015a). Extension of use of extracts of rosemary (E 392) in fat-based spreads. Retrieved from
1234 https://efsa.onlinelibrary.wiley.com/doi/abs/10.2903/j.efsa.2015.4090.
1235EFSA. (2015b). Scientific Opinion on the re-evaluation of ascorbic acid (E 300), sodium ascorbate (E
1236301) and calcium ascorbate (E 302) as food additives. EFSA Journal, 13(5), 4087.
1237doi:doi:10.2903/j.efsa.2015.4087
1238EFSA, T. r.-E. v. o. E. s. f. a. A. i. S., 2018. https://www.efsa.europa.eu/en/press/news/120130b (2012).
1239The re-‘E’valuation of Europe’s food additives. Retrieved from
1240 https://www.efsa.europa.eu/en/press/news/120130b
1241EMA. (2000). Committee for Proprietary Medicinal Products (CPMP). Points to consider on switching
1242between superiority and non-inferiority.
1243EMA. (2005). Committee on Herbal Medicinal Products. Assessment report on Symphytum officinale L.,
1244radix., EMA/HMPC/572844/2009.

521245Erdemoglu, N., Ozkan, S., & Tosun, F. (2007). Alkaloid profile and antimicrobial activity of Lupinus
1246angustifolius L. alkaloid extract. Phytochemistry Reviews, 6(1), 197-201.
1247Frost, R., MacPherson, H., & O'Meara, S. M. (2013). A critical scoping review of external uses of
1248comfrey (Symphytum spp.). Complementary Therapies in Medicine, 21(6), 724-745.
1249doi:10.1016/j.ctim.2013.09.009
1250Gafar, M., Dumitriu, H., Dumitriu, S., & Guti, L. (1989). Apiphytotherapeutic original preparations in the
1251treatment of chronic marginal parodontopathies. A clinical and microbiological study. Revista de
1252chirurgie, oncologie, radiologie, o. r. l., oftalmologie, stomatologie. Seria: Stomatologie, 36, 91-
125398.
1254Giannetti, B. M., Staiger, C., Bulitta, M., & Predel, H.-G. (2009). Efficacy and safety of comfrey root
1255extract ointment in the treatment of acute upper or lower back pain: results of a double-blind,
1256randomised, placebo controlled, multicentre trial. British journal of sports medicine,
1257bjsports58677.
1258Gomes, M. F. P. L., Massoco, C. O., Xavier, J. G., & Bonamin, L. V. (2010). Comfrey (Symphytum
1259officinale L.) and experimental hepatic carcinogenesis: a short-term carcinogenesis model study.
1260eCAM, 7, 197–202.
1261Grabias, B., & Swiatek, L. (1998). Phenolic acids in Symphytum officinale L. Pharmaceutical and
1262Pharmacological Letters, 8, 81-83.
1263Grieve, B. (1984). A Modern Herbal: Penguin.
1264Grigore, A., Pirvu, L., Bubueanu, C., Minerva, P., & Rasit, I. (2015). Influence of chemical composition
1265on the antioxidant and anti-inflammatory activity of Rosmarinus officinalis extracts. Rom.
1266Biotechnol. Lett., 20(1), 10047- 10054.
1267Grube, B., Grünwald, J., Krug, L., & Staiger, C. (2007). Efficacy of a comfrey root (Symphyti offic.
1268radix) extract ointment in the treatment of patients with painful osteoarthritis of the knee: results
1269of a double-blind, randomised, bicenter, placebo-controlled trial. Phytomedicine, 14(1), 2-10.
1270Grünwald, J., Bitterlich, N., Nauert, C., & Schmidt, M. (2010). Anwendung und Verträglichkeit von
1271Beinwellcreme (Symphyti herba) bei Kindern mit akuten stumpfen Traumen. Zeitschrift für
1272Phytotherapie, 31(01), 61-66.
1273Haaß, D., Abou-Mandour, A. A., Blaschek, W., Franz, G., & Czygan, F. C. (1991 ). he influence of
1274phytohormones on growth, organ differentiation and fructan production in callus of Symphytum
1275officinale L. Plant Cell Rep., 10, 421-424.
1276Hacıoğlu, B. T., & Erik, S. (2011). Phylogeny of Symphytum L.(Boraginaceae) with special emphasis on
1277Turkish species. African Journal of Biotechnology, 10(69), 15483-15493.
1278Häkkinen, S. H., Kärenlampi, S. O., Mykkänen, H. M., Heinonen, I. M., & Törrönen, A. R. (2000).
1279Ellagic acid content in berries: Influence of domestic processing and storage. European Food
1280Research and Technology, 212(1), 75-80. doi:10.1007/s002170000184
1281Harris, P. J. C., Grove, C. G., & Havard, A. J. (1989). In vitro propagation of Symphytum species.
1282Scientia Horticulturae, 40(4), 275-281. doi: https://doi.org/10.1016/0304-4238(89)90101-5
1283Hiermann, A., & Writzel, M. (1998). Antiphlogistic glycopeptide from the roots of Symphytum
1284officinale. Pharmaceutical & Pharmacological Letters, 8(4), 154-157.
1285Horinouchi, C. D., & Otuki, M. F. (2013). Botanical briefs: comfrey (Symphytum officinale). Cutis,
128691(5), 225-228.
1287Huxley, A. (1992). The New RHS Dictionary of Gardening: MacMillan Press.
1288Janes, D., Kalamar, B., & Kreft, S. (2012 ). Improved method for isolation of lycopsamine from roots of
1289comfrey (Symphytum officinale). Nat Prod Commun, 7, 861-862.
1290Janeš, D., & Kreft, S. (2014 ). TLC densitometric method for screening of lycopsamine in comfrey root
1291(Symphytum officinale L.) extracts using retrorsine as a reference compound. Acta Pharm., 64,
1292503-508.
1293Juhl, V. M. (1978). Liste over vaertplanter for blad nematoden Aphelenchoides ritzemabosi. Ugeskrift for
1294Agronomer, Hortonomer, Forstkandidater Og Licentiater, 123, 183-186.

531295Kapil, A. (2005). The challenge of antibiotic resistance: need to contemplate. Indian J Med Res., 121(2),
129683-91.
1297Karavaev, V. A., Solntsev, M. K., Iurina, T. P., Iurina, E. V., Poliakova, I. B., & Kuznetsov, A. M.
1298(2001a). Antifungal activity of aqueous extracts from the leaf of cowparsnip and comfrey. Izv
1299Akad Nauk Ser Biol, 4(4), 435-441.
1300Karavaev, V. A., Solntsev, M. K., Iurina, T. P., Iurina, E. V., Poliakova, I. B., & Kuznetsov, A. M.
1301(2001b). [Antifungal activity of aqueous extracts from the leaf of cowparsnip and comfrey]. Izv
1302Akad Nauk Ser Biol(4), 435-441.
1303Kartal, M., Kurucu, S., & Choudary, M. I. (2001). Antifungal activities of different extracts and
1304echimidine-N-oxide from Symphytum sylvaticum Boiss. subsp. sepulcrale (Boiss. & Bal.) Greuter
1305& Burdet var. Sepulcrale Turkish Journal of Medical Sciences, 31(6), 487-492.
1306Kim, N. C., Oberlies, N. H., Brine, D. R., Handy, R. W., Wani, M. C., & Wall, M. E. (2001). Isolation of
1307symlandine from the roots of common comfrey (Symphytum officinale) using countercurrent
1308chromatography. J Nat Prod, 64, 251-253.
1309Knaak, N., Dias da Silva, L., Finger Andreis, T., & Mariana Fiuza, L. (2013). Chemical characterization
1310and anti-fungal activity of plant extracts and essential oils on the Bipolaris oryzae and Gerlachia
1311oryzae phytopathogens. Australasian Plant Pathology, 42, 469-475.
1312Knight, K. W., Barber, C. J., & Page, G. D. (1997). Plant-parasitic Nematodes of New Zealand Recorded
1313by Host Association. Journal of nematology, 29(4S), 640-656.
1314Koll, R., Buhr, M., Dieter, R., Pabst, H., Predel, H.-G., Petrowicz, O., . . . Staiger, C. (2004). Efficacy and
1315tolerance of a comfrey root extract (Extr. Rad. Symphyti) in the treatment of ankle distorsions:
1316results of a multicenter, randomized, placebo-controlled, double-blind study. Phytomedicine,
131711(6), 470-477.
1318Kommission, E. (1990). Monographie Symphyti radix (Beinwellwurzel). Bundesanzeiger, 318.
1319Kruse, L. H., Stegemann, T., Sievert, C., & Ober, D. (2017). Identification of a Second Site of
1320Pyrrolizidine Alkaloid Biosynthesis in Comfrey to Boost Plant Defense in Floral Stage(). Plant
1321Physiol, 174(1), 47-55. doi:10.1104/pp.17.00265
1322Kucera, M., Barna, M., Horácek, O., Kálal, J., Kucera, A., & Hladíkova, M. (2005). TopicalSymphytum
1323herb concentrate cream against myalgia: A randomized controlled double-blind clinical study.
1324Advances in therapy, 22(6), 681-692.
1325Kučera, M., Barna, M., Horáček, O., Kováriková, J., & Kučera, A. (2004). Efficacy and safety of
1326topically applied Symphytum herb extract cream in the treatment of ankle distortion: Results of a
1327randomized controlled clinical double-blind study. Wiener Medizinische Wochenschrift, 154(21-
132822), 498-507.
1329Kucera, M., Kálal, J., & Polesna, Z. (2000). Effects ofSymphytum ointment on muscular symptoms and
1330functional locomotor disturbances. Advances in therapy, 17(4), 204-211.
1331Kurucu, S., Kartal, M., Choudary, M. I., & Topcu, G. (2002). Pyrrolizidine Alkaloids from Symphytum
1332sylvaticum Boiss. subsp. sepulcrale. (Boiss. & Bal.) Greuter & Burdet var. sepulcrale and
1333Symphytum aintabicum Hub. – Mor. & Wickens. Turkish Journal of Chemistry, 26, 195-199.
1334Laslett, L., Quinn, S., Darian-Smith, E., Kwok, M., Fedorova, T., Körner, H., . . . Jones, G. (2012).
1335Treatment with 4Jointz reduces knee pain over 12 weeks of treatment in patients with clinical
1336knee osteoarthritis: a randomised controlled trial. Osteoarthritis and cartilage, 20(11), 1209-
13371216.
1338Lee, N. K., & Paik, H. D. (2016). Status, Antimicrobial Mechanism, and Regulation of Natural
1339Preservatives in Livestock Food Systems. Korean Journal for Food Science of Animal Resources,
134036(4), 547-557
1341Lewis, K., & Ausubel, F. (2006). Prospects for plant derived antibacterials. Nat Biotechnol, 24(12), 1504-
13421507.

541343Liu, C., Chang, J., Zhang, L., Zhang, J., & Li, S. (2012). Purification and antioxidant activity of a
1344polysaccharide from bulbs of Fritillaria ussuriensis Maxim. Int J Biol Macromol, 50(4), 1075-
13451080.
1346Liu, F., Wan, S. Y., Jiang, Z., Li, S. F. Y., Ong, E. S., & Osorio, J. C. C. (2009). Determination of
1347pyrrolizidine alkaloids in comfrey by liquid chromatography–electrospray ionization mass
1348spectrometry. Talanta, 80, 916–923.
1349Ma, C., Liu, Y., Zhu, L., Ji, H., Song, X., Guo, H., & Yi, T. (2018). Determination and regulation of
1350hepatotoxic pyrrolizidine alkaloids in food: A critical review of recent research. Food and
1351Chemical Toxicology. . Food and Chemical Toxicology.
1352MacDonald, R., & Reitmeier, C. (2017). Understanding Food Systems: Agriculture, Food Science, and
1353Nutrition in the United States. US: Academic Press.
1354MacKay, D., & Miller, A. L. (2003). Nutritional support for wound healing. Altern Med Rev, 8(4), 359-
1355377.
1356Matcovschi, C., Calistru, Z., Cojocaru, M., & Vaˆlcu, S. (1995). Symphytum officinale (L) gaertn — A
1357prospective hepatoprotective and hepatoregenerative plant. Pharmacological Research, 31, 91.
1358doi: https://doi.org/10.1016/1043-6618(95)86625-6
1359Matthew, J., & Leach, R. N. (2004). A critical review of natural therapies in wound management. Ostomy
1360Wound Manage, 50, 36–51.
1361Mazzocchi, A., & Montanaro, F. (2012). Observational study of the use of Symphytum 5CH in the
1362management of pain and swelling after dental implant surgery. Homeopathy, 101(4), 211-216.
1363doi:10.1016/j.homp.2012.07.002
1364Mei, N., Guo, L., Fu, P. P., Fuscoe, J. C., Luan, Y., & Chen, T. (2010). Metabolism, Genotoxicity, annd
1365Carcinogenicity of Comfrey. Journal of Toxicology and Environmental Health, Part B, 13(7-8),
1366509-526. doi:10.1080/10937404.2010.509013
1367Melkumova, Z. V., Telezhenetskaya, M. V., Yunusov, S. Y., & Manko, I. V. (1974). Refinement of the
1368structure of asperumine. Chemistry of Natural Compounds, 10, 483–485.
1369Merlani, M., Barbakadze, V., Amiranashvili, L., Gogilashvili, L., Yannakopoulou, E., Papadopoulos, K.,
1370& Chankvetadze, B. (2010). Enantioselective synthesis and antioxidant activity of 3-(3,4-
1371dihydroxyphenyl)-glyceric acid–basic monomeric moiety of a biologically active polyether from
1372Symphytum asperum and S. caucasicum. Chirality, 22(8), 717-725. doi:10.1002/chir.20823
1373Mohammad, F. V., Noorwala, M., Ahmad, V. U., & Sener, B. (1995 ). A bidesmosidic hederagenin
1374hexasaccharide from the roots of Symphytum officinale. Phytochemistry, 40, 213-218.
1375Morteza-Semnani, K., Amin, G., Shidfar, M., Hadizadeh, H., & Shafiee, A. (2003). Antifungal activity of
1376the methanolic extract and alkaloids of Glaucium oxylobum. Fitoterapia, 74(5), 493-496.
1377Mroczek, T., Ndjoko-Ioset, K., Głowniak, K., Mietkiewicz-Capała, A., & Hostettmann, K. (2006).
1378Investigation of Symphytum cordatum alkaloids by liquid–liquid partitioning, thin-layer
1379chromatography and liquid chromatography–ion-trap mass spectrometry. Analytica Chimica
1380Acta, 566 157–166.
1381Mulkijanyan, K., Barbakadze, V., Novikova, Z., Sulakvelidze, M., Gogilashvili, L., Amiranashvili, L., &
1382Merlani, M. (2009). Burn healing compositions from Caucasian species of comfrey (Symphytum
1383L.). Bull Georg Natl Acad Sci, 3(3), 114-117.
1384Mutterlein, R., & Arnorld, C. G. (1993). Investigations concerning the content and the pattern of
1385pyrrolizidine alkaloids in Symphytum officinale L. (comfrey). Pharm Ztg Wiss, 138, 119-125.
1386Neagu, E., Moroeanu, V., & Radu, G. (2008). Concentration of Symphytum officinale extracts with
1387cytostatic activity by tangential flow ultrafiltration. Roumanian Biotechnological Letters 13(6),
13884008-4013.
1389Neagu, E., Paun, G., & Radu, G. (2010). Antioxidant capacity of some Symphytum officinalis extracts
1390processed by ultrafiltration. Romanian Biotechnological Letters, 15(4), 5505-5511.
1391Neagu, E., Paun, G., & Radu, L. G. (2011 ). Phytochemical study of some Symphytum officinalis extracts
1392concentrated by membranous procedures U.P.B. Sci. Bull., Series B, 73, 65-74.

551393NEAGU, E., PĂUN, G., & RADU, L. G. (2011). Phytochemical study of some Symphytum officinalis
1394extracts concentrated by membranous procedures. Scientific Bulletin-University Politehnica
1395Buchacharest, 3-7.
1396Neagu, E., Roman, G. P., & Radu, G. L. (2010). Antioxidant capacity of some Symphytum officinalis
1397extracts processed by ultrafiltration. Romanian Biotechnological Letters, 15(4), 5505-5511.
1398Neuman, M. G., Cohen, L., Opris, M., Nanau, R. M., & Jeong, H. (2015). Hepatotoxicity of pyrrolizidine
1399alkaloids. Journal of Pharmacy & Pharmaceutical Sciences, 18(4), 825-843.
1400Nossa González, D. L., Pérez, T., Verónica, Y., Núñez, R., & Elías, W. (2016). Determination of
1401polyphenols and antioxidant activity of polar extracts of comfrey (Symphytum officinale L).
1402Revista Cubana de Plantas Medicinales, 21(2), 125-132.
1403Oberbaum, M., Yakovlev, E., Kaufman, D., & Shoshan, S. (1994). Effect of Arnica montana and
1404Symphytum officinalis on bone healing in guinea pigs. British Homeopathic Journal, 83(2), 90.
1405doi:10.1016/S0007-0785(94)80017-0
1406Oberlies, N. H., Kim, N. C., Brine, D. R., Collins, B. J., Handy, R. W., Sparacino, C. M., . . . Wall, M. E.
1407(2004 ). Analysis of herbal teas made from the leaves of comfrey (Symphytum officinale):
1408reduction of N-oxides results in order of magnitude increases in the measurable concentration of
1409pyrrolizidine alkaloids. Public Health Nutr., 7, 919-924.
1410Oktay, M., Yildirim, A., Bilaloglu, V., & Gülçin, I. (2007). Antioxidant activity of different parts of isgin
1411(Rheum ribes L.). Asian Journal of Chemistry, 19(4), 3047.
1412Onduso, S. O. (2014). Determination of levels of pyrrolizidine alkaloids in Symphytum asperum Lepech
1413growing in selected parts of Keniya. (Master of Science (Chemistry)), Kenyatta University.
1414Onofre, N. A., Magalhães, L. P. M., Amaral, J. P. D., Yara, R., Lima, C. S. A., & Sena, K. X. F. R.
1415(2016). Antimicrobial activity of plants used in manufacture of traditional phytotherapics in
1416community centres of Recife metropolitan region. Experimental Pathology and Health Sciences,
14178(3), 23-24.
1418Orescanin, V. (2016). Treatment of atopic dermatitis in children with Bioapifit® anti-inflammatory herbal
1419ointment – a preliminary study. IJRDO-Journal of Biological Science, 2, 1-17.
1420Orescanin, V. (2016). Treatment of pressure ulcers with Bioapit® wound healing herbal ointment – a
1421preliminary study. IJRDO-Journal of Biological Science, 2, 1-14.
1422Orescanin, V., Guštek Štefica, F., & Krivak Bolanca, I. (2015). Development and Aplication of New
1423Herbal Pessaries for the Treatment of Squamous Endocervical Metaplasia. Indian Journal of
1424Applied Research, 5, 176-182.
1425Pabst, H., Schaefer, A., Staiger, C., Junker‐Samek, M., & Predel, H. G. (2013). Combination of comfrey
1426root extract plus methyl nicotinate in patients with conditions of acute upper or low back pain: a
1427multicentre randomised controlled trial. Phytotherapy Research, 27(6), 811-817.
1428Paun, G., Neagu, E., Litescu, S. C., Rotinberg, P., & Radu, G. L. (2012). Application of membrane
1429processes for the concentration of Symphytum officinale and Geranium robertianum extracts to
1430obtain compounds with high anti-oxidative activity. Journal of the Serbian Chemical Society,
143177(9).
1432Pawlowski, B. (1972). Symphytum L. and Procopiania Gusul. In T. Tutin, Heywood V, Burges H, Moore
1433NA, Valentine DM, Walters DH, Webb SM (Ed.), Flora of Europea (Vol. 3, pp. 103-106).
1434Cambridge: Cambridge University Press.
1435Peruzzi, L., Garbari, F., & Bottega, S. (2001). Symphytum tanaicense (Boraginaceae) new for the Italian
1436flora. Willdenowia, 31(1), 33-41. doi:10.3372/wi.31.31102
1437Petersen, G., Lorkowski, G., Kasper, F., Gottwald, R., & Lücker, P. (1993). Anti-inflammatory activity of
1438a pyrrolizidine alkaloid-free extract of roots of Symphytum officinale in humans. Planta Medica,
143959(S 1), A703-A704.
1440Petkeviciute, Z., Savickiene, N., Savickas, A., Bernatoniene, J., Simaitiene, Z., Kalveniene, Z., . . .
1441Mekas, T. A. (2010). Urban ethnobotany study in Samogitia region, Lithuania. Journal of
1442Medicinal Plants Research, 4(1), 064-071.

561443Pielesz, A. (2012). Vibrational spectroscopy and electrophoresis as a "golden means" in monitoring of
1444polysaccharides in medical plant and gels. Spectrochimica Acta – Part A: Molecular and
1445Biomolecular Spectroscopy, 93, 63-69. doi:10.1016/j.saa.2012.03.003
1446Pileggi, M., Raiman, P. M., Micheli, A., Beatriz, S., & Bobalto, V. (2002). Antimicrobial Action and
1447Endophytic interaction in Symphytum officinale L. Publicatio UEPG, 8(1), 47-55.
1448Prakash, A. M., Sharifi-Rad, M., Shariati, M., Mabkhot, Y., Al-Showiman, S., Rauf, A., . . . Sharifi-Rad,
1449J. (2018). Bioactive compounds and health benefits of edible Rumex species-A review. Cellular
1450and molecular biology (Noisy-le-Grand, France), 64(8), 27-34.
1451Prakash, A. S., Pereira, T. N., Reilly, P. E., & Seawright, A. A. (1999). Pyrrolizidine alkaloids in human
1452diet. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 443(1), 53-67.
1453Prakash Mishra, A., Saklani, S., Salehi, B., Parcha, V., Sharifi-Rad, M., Milella, L., . . . Srivastava, M.
1454(2018). Satyrium nepalense, a high altitude medicinal orchid of Indian Himalayan region:
1455chemical profile and biological activities of tuber extracts. Cellular and molecular biology
1456(Noisy-le-Grand, France), 64(8), 35-43.
1457Predel, H.-G., Giannetti, B., Koll, R., Bulitta, M., & Staiger, C. (2005). Efficacy of a Comfrey root extract
1458ointment in comparison to a Diclo-fenac gel in the treatment of ankle distortions: Results of an
1459observer-blind, randomized, multicenter study. Phytomedicine, 12(10), 707-714.
1460Radermacher, J., Jentsch, D., Scholl, M., Lustinetz, T., & Frolich, J. (1991). Diclofenac concentrations in
1461synovial fluid and plasma after cutaneous application in inflammatory and degenerative joint
1462disease. British journal of clinical pharmacology, 31(5), 537-541.
1463Repetto, M., & Llesuy, S. (2002). Antioxidant properties of natural compounds used in popular medicine
1464for gastric ulcers. Brazilian journal of medical and biological research, 35(5), 523-534.
1465Riess, W., Schmid, K., Botta, L., Kobayashi, K., Moppert, J., Schneider, W., . . . Tomasi, M. (1986). The
1466percutaneous absorption of diclofenac. Arzneimittel-Forschung, 36(7), 1092-1096.
1467Riet-Correa, F., Medeiros, R., Tokarnia, C., & Dobereiner, J. (2007). Toxic plants for livestock in Brazil:
1468Economic impact, toxic species, control measures and public health implications. In T. W. a. J. P.
1469s. KE Panter (Ed.), Poisonous Plants: Global Research and Solutions (pp. 2-14). Wallingford,
1470UK: CABI Press.
1471Robinson, R. G. (1983). Comfrey—A Controversial Crop. Retrieved from St. Paul, Minnesota:
1472Rocha, R., Eleutério da Luz, D., Engels, C., Pileggi, S., de Souza Jaccoud Filho, D., Matiello, R., &
1473Pileggi, M. (2009). Selection of endophytic fungi from comfrey (Symphytum officinale L.) for in
1474vitro biological control of the phytopathogen Sclerotinia sclerotiorum (Lib.). Brazilian Journal of
1475Microbiology, 40, 73-78.
1476Rode, D. (2002). Comfrey toxicity revisited. Trends Pharmacol Sci, 23(11), 497-499.
1477Rodrigues Oliveira, P., Ramos Santos, F., Ferreira Duarte, E., Silva Guimarães, G., Sartori Carvalho
1478Mattos, N., & Minafra, C. (2016). Symbiotics and Aloe vera and Symphytum officinale extracts
1479in broiler feed. Semina: Ciencias Agrarias, 37(4Supl1), 2677. doi:10.5433/1679-
14800359.2016v37n4Supl1p2677
1481Roeder, E. (1995). Medicinal plants in Europe containing pyrrolizidine alkaloids. Pharmazie, 50, 83-98.
1482Roeder, l. E., Bourauel, T., & Neuberger, V. (1992). Symviridine, a new pyrrolizidine alkaloid from
1483Symphytum species. Phytochemistry, 31, 4041-4042.
1484Salehi, B., Valussi, M., Jugran, A. K., Martorell, M., Ramírez-Alarcón, K., Stojanović-Radić, Z. Z., . . .
1485Sharifi-Rad, M. (2018). Nepeta Species: From Farm to Food applications and Phytotherapy.
1486Trends in Food Science & Technology, 80, 104-122.
1487Savic, V., Nikolic, V., Stanojevic, L., Ilic, D., & Stankovic, B. (2012). Extraction kinetics and antioxidant
1488activity of an aqueous extract from comfrey root (Symphytum officinale L.). Advanced
1489technologies, 1(2), 41–47.
1490Savić, V. L., Nikolić, V. D., Arsić, I. A., Stanojević, L. P., Najman, S. J., Stojanović, S., & Mladenović-
1491Ranisavljević, I. (2015). Comparative study of the biological activity of allantoin and aqueous
1492extract of the comfrey root. Phytother Res, 29, 1117-1122.

571493Savić, V. L., Nikolić, V. D., Arsić, I. A., Stanojević, L. P., Najman, S. J., Stojanović, S., & Mladenović-
1494Ranisavljević, I. I. (2015). Comparative Study of the Biological Activity of Allantoin and
1495Aqueous Extract of the Comfrey Root. Phytotherapy Research, 29(8), 1117-1122.
1496doi:doi:10.1002/ptr.5356
1497Savić, V. L., Savić, S. R., Nikolić, V. D., Nikolić, L. B., Najman, S. J., Lazarević, J. S., & Đorđević, A. S.
1498(2015). The identification and quantification of bioactive compounds from the aqueous extract of
1499comfrey root by UHPLC-DAD-HESI-MS method and its microbial activity. Hemijska Industrija,
150069(1), 1-8.
1501Schmelzer, G., & Gurib-Fakim, A. (2008). Plant Resources of Tropical Africa: Medicinal plants 1.
1502Netherlands: Backhuys Publishers CTA.
1503Schmid, K., Ivemeyer, S., Vogl, C., Klarer, F., Meier, B., Hamburger, M., & Walkenhorst, M. (2012).
1504Traditional use of herbal remedies in livestock by farmers in 3 Swiss cantons (Aargau, Zurich,
1505Schaffhausen). Forsch Komplementmed, 19(3), 125-136. doi:10.1159/000339336
1506Schmidtke-Schrezenmeier, G. (1992). The efficacy of a phytotherapeutic agent in the treatment of non-
1507activated osteoarthritis of the knee [Behandlung der nichtaktivierten Gonarthrose: Besserung
1508durch ein Phytotherapeutikum]. Therapiewoche, 42, 1322-1325.
1509Settanni, L., & Corsetti, A. (2008). Application of bacteriocins in vegetable food biopreservation.
1510International Journal of Food Microbiology, 121(2), 123-138.
1511Shang, H., Zhou, H., Duan, M., Li, R., Wu, H., & Lou, Y. (2018). Extraction condition optimization and
1512effects of drying methods on physicochemical properties and antioxidant activities of
1513polysaccharides from comfrey (Symphytum officinale L.) root. Int J Biol Macromol, 112, 889-
1514899. doi:10.1016/j.ijbiomac.2018.01.198
1515Sharifi-Rad, J., Sharifi-Rad, M., Salehi, B., Iriti, M., Roointan, A., Mnayer, D., . . . Afshari, A. (2018). In
1516vitro and in vivo assessment of free radical scavenging and antioxidant activities of Veronica
1517persica Poir. Cellular and molecular biology (Noisy-le-Grand, France), 64(8), 57-64.
1518Sharifi-Rad, J., Soufi, L., Ayatollahi, S., Iriti, M., Sharifi-Rad, M., Varoni, E. M., . . . Kuhestani, K.
1519(2016). Anti-bacterial effect of essential oil from Xanthium strumarium against shiga toxin-
1520producing Escherichia coli. Cellular and Molecular Biology, 62(9), 69-74.
1521Sharifi-Rad, J., Tayeboon, G., Niknam, F., Sharifi-Rad, M., Mohajeri, M., Salehi, B., & Iriti, M. (2018).
1522Veronica persica Poir. extract-antibacterial, antifungal and scolicidal activities, and inhibitory
1523potential on acetylcholinesterase, tyrosinase, lipoxygenase and xanthine oxidase. Cellular and
1524molecular biology (Noisy-le-Grand, France), 64(8), 50-56.
1525Sharifi-Rad, M., Fokou, P., Sharopov, F., Martorell, M., Ademiluyi, A., Rajkovic, J., . . . Sharifi-Rad, J.
1526(2018). Antiulcer Agents: From Plant Extracts to Phytochemicals in Healing Promotion.
1527Molecules (Basel, Switzerland), 23(7).
1528Sharifi-Rad, M., Nazaruk, J., Polito, L., Morais-Braga, M., Rocha, J., Coutinho, H., . . . Del, M. M. C.
1529(2018). Matricaria genus as a source of antimicrobial agents: From farm to pharmacy and food
1530applications. Microbiological research, 215, 76-88.
1531Singh, H., Du, J., Singh, P., & Yi, T. H. (2018). Role of green silver nanoparticles synthesized from
1532Symphytum officinale leaf extract in protection against UVB-induced photoaging. Journal of
1533Nanostructure in Chemistry, 8(3), 359-368. doi:10.1007/s40097-018-0281-6
1534Slobodníková, L., KoSt'álová, D., Labudová, D., Kotulová, D., & Kettmann, V. (2004). Antimicrobial
1535activity of Mahonia aquifolium crude extract and its major isolated alkaloids. Phytotherapy
1536Research: An International Journal Devoted to Pharmacological and Toxicological Evaluation of
1537Natural Product Derivatives, 18(8), 674-676.
1538Smith, D. B., & Jacobson, B. H. (2011). Effect of a blend of comfrey root extract (Symphytum officinale
1539L.) and tannic acid creams in the treatment of osteoarthritis of the knee: randomized, placebo-
1540controlled, double-blind, multiclinical trials. Journal of chiropractic medicine, 10(3), 147-156.

581541Smith, N., Shin, D. B., Brauer, J. A., Mao, J., & Gelfand, J. M. (2009). Use of complementary and
1542alternative medicine among adults with skin disease: results from a national survey. J Am Acad
1543Dermatol, 60(3), 419-425. doi:10.1016/j.jaad.2008.11.905
1544Sowa, I., Paduch, R., Strzemski, M., Zielińska, S., Rydzik-Strzemska, E., Sawicki, J., . . . Latalski, M.
1545(2018). Proliferative and antioxidant activity of Symphytum officinale root extract. Natural
1546Product Research, 32(5), 605-609
1547Sowa, I., Paduch, R., Strzemski, M., Zielińska, S., Rydzik-Strzemska, E., Sawicki, J., . . . Wójciak-
1548Kosior, M. (2018). Proliferative and antioxidant activity of Symphytum officinale root extract.
1549Natural Product Research, 32(5), 605-609. doi:10.1080/14786419.2017.1326492
1550Staiger, C. (2012). Comfrey: a clinical overview. Phytother Res, 26(10), 1441-1448. doi:10.1002/ptr.4612
1551Staiger, C. (2013). Comfrey root: from tradition to modern clinical trials. Wien Med Wochenschr, 163(3-
15524), 58-64.
1553Štepán, J., Ehrlichova, J., & Hladikova, M. (2014). Therapieergebnisse und Anwendungssicherheit von
1554Symphytum-Herba-Extrakt-Creme in der Behandlung von Dekubitus. Zeitschrift für
1555Gerontologie und Geriatrie, 47(3), 228-235.
1556Stickel, F., & Seitz, H. K. (2000). The efficacy and safety of comfrey. Public Health Nutrition, 3(4a),
1557501-508.
1558Sumathi, S. (2016). Antibacterial Activity of the plant extract of Symphytum officinale L. against selected
1559pathogenic bacteria. International Journal of Research in Pharmaceutical Sciences, 2(1), 92-94
1560Sumathi, S., Kumar, S. S., Bai, A., & Glory, L. (2011). Evaluation of Phytochemical Constituents and
1561Antibacterial Activities of Symphytum officinale L. . Journal of Pure and Applied Microbiology,
15625(1), 323-328.
1563Tamariz, J., Burgueño-Tapia, E., Vázquez, M. A., & Delgado, F. (2018). Pyrrolizidine Alkaloids The
1564Alkaloids: Chemistry and Biology (Vol. 80, pp. 1-314).
1565Tarıkahya, B. (2010). The Revision of Turkish Symphytum L. (Boraginaceae) Genus. (Ph.D.), Hacettepe
1566University, Ankara.
1567Teynor, T. M., Putnam, D. H., Doll, J. D., Kelling, K. A., Oelke, E. A., Undersander, D. J., & Oplinger,
1568E. S. (1992). Comfrey – Alternative Field Crops Manual. Minnesota: Minnesota Extension
1569Service, University of Minnesota.
1570Thoresen, E. M. (2013). Symphytum officinale Common comfrey. (Ph.D.), Szent Istvan University,
1571Budapest
1572Thring, T. S., Hili, P., & Naughton, D. P. (2009). Anti-collagenase, anti-elastase and anti-oxidant
1573activities of extracts from 21 plants. BMC complementary and alternative medicine, 9(1), 27.
1574Trifan, A., Opitz, S. E. W., Josuran, R., Grubelnik, A., Esslinger, N., Peter, S., . . . Wolfram, E. (2018). Is
1575comfrey root more than toxic pyrrolizidine alkaloids? Salvianolic acids among antioxidant
1576polyphenols in comfrey (Symphytum officinale L.) roots. Food and Chemical Toxicology, 112,
1577178-187.
1578Tsutomu Furuya, & Hikichi, M. (1971). Alkaloids and triterpenoids of Symphytum officinale.
1579Phytochemistry, 10, 2217-2220.
1580Ulubelen, A., & Öcal, F. (1977). Alkaloids and other compounds of Symphytum tuberosum.
1581Phytochemistry, 16, 499-500.
1582Üstün Alkan, F., Anlas, C., Ustuner, O., Bakırel, T., & Sari, A. (2014). Antioxidant and proliferative
1583effects of aqueous and ethanolic extracts of Symphytum officinale on 3T3 Swiss albino mouse
1584fibroblast cell line. Asian Journal of Plant Science and Research, 4(4), 62-68.
1585Vostinaru, O., Conea, S., Mogosan, C., Toma, C., Borza, C., & Vlase, L. (2017). Anti-inflammatory and
1586antinociceptive effect of Symphytum officinale root. Romanian Biotechnological Letters.
1587Wedzicha, B. L. (2003). PRESERVATIVES | Classifications and Properties., 4773-4776.
1588doi:10.1016/B0-12-227055-X/00969-X
1589WHO. (2014). WHO Traditional Medicine Strategy 2014-2023. World Health Organization Retrieved
1590from http:llapps.who.int7irislbitstream710665l92455l1l9789241506090eng.pdf?ua.

591591WHO. (2015). WHO estimates of the global burden of foodborne diseases. Geneva: World Health
1592Organization.
1593Woods-Panzaru, S., Nelson, D., McCollum, G., Ballard, L. M., Millar, B. C., Maeda, Y., . . . Moore, J. E.
1594(2009). An examination of antibacterial and antifungal properties of constituents described in
1595traditional Ulster cures and remedies. The Ulster Medical Journal, 78(1), 13-15.
1596Wuilloud, J. C., Gratz, S. R., Gamble, B. M., & Wolnik, K. A. (2004). Simultaneous analysis of
1597hepatotoxic pyrrolizidine alkaloids and N-oxides in comfrey root by LC-ion trap mass
1598spectrometry. Analyst, 129(2), 150-156.
1599Yunusova, S. G., Lyashenko, S. S., Fedorov, N. I., Fedorov, N. I., & Denisenko, O. N. (2017). Lipids and
1600lipophilic constituents of comfrey (Symphytum officinale L.) seeds. Pharmaceutical Chemistry
1601Journal, 50, 728-731.

60Table 1. A literature report on the main constitutions of Symphytum plants from different geographical regions.
Species Anatomical part Main components Reference
S. asperum Roots Intermedine N-oxides, lycopsamine N-oxides, 7-acetyl symlandine, 7-acetyl
symviridine, myoscorpine, symphytine, echimidine(E. Roeder, 1995; l. E.
Roeder et al., 1992)
S. asperum Roots Echimidine, 7- acetyl lycopsamine, 3‘-acetyl lycopsamine, triangularine and
heliosupine(Onduso, 2014)
S. asperum Roots Anticomplementary dihydroxycinnamate-derived polymer (V.V. Barbakadze et al.,
2000)
S. asperum and S.
caucasicumRoots Poly[oxy-1-carboxy-2-(3,4- dihydroxyphenyl)ethylene], poly[3-(3,4-
dihydroxyphenyl)glyceric acid(V. Barbakadze et al.,
2005)
S. asperum and S.
caucasicumRoots and stems Poly[oxy-1-carboxy-2-(3,4-dihydroxyphenyl) ethylene] (V.V. Barbakadze et al.,
2007)
S. caucasicum Bieb. Roots Asperumine, echimidine N-oxide, echinatine, and lasiocarpine (Melkumova et al., 1974;
E. Roeder, 1995)
S. cordatum (L.) W.K Roots Echimidine N-oxide (three diasteroisomers), 7-sarracinyl-9-
viridiflorylretronecine (two diasteroisomers), echimidine (two
diasteroisomers), lycopsamine (two diasteroisomers), dihydroechinatine N-
oxide, dihydroheliospathuline N-oxide, lycopsamine N-oxide (three
diasteroisomers), 7-acetyl lycopsamine N-oxide, symphytine N-oxide (two
diasteroisomers) and 2,3-epoxyechiumine N-oxide(Mroczek et al., 2006)
S. officinale Roots Intermedine, lycopsamine, intermedine N-oxide, lycopsamine N-oxide, 7-
acetyl intermedine, 7-acetyl lycopsamine, 7- acetyl intermedine N-oxide, 7-
acetyl lycopsamine N-oxide, uplandicine N-oxide, myoscorpine, echiumine,
symphytine, symviridine, myoscorpine N-oxide, echiumine N-oxide,
symphytine N-oxide, symviridine N-oxide, heliosupine, asperumine,
heliosupine N-oxide, asperumine N-oxide (Avula et al., 2015)
S. officinale – Lycopsamine, echimidine, lasiocarpine (F. Liu et al., 2009)
S. officinale Roots Symlandine, symphytine, echimidine (Kim et al., 2001)
S. officinale Roots Lycopsamine (Janes et al., 2012 ; Janeš
& Kreft, 2014 )
S. officinale 7-acetyl intermedine, 7-acetyl lycopsamine (Brauchli et al., 1982)

61S. officinale, S. asperum,
S. x uplandicumRoots Symviridine (l. E. Roeder et al., 1992)
S. officinale, S. x
uplandicumRoots Intermedine, intermedine N-oxide, 7-acetyl lycopsamine, acetyl lycopsamine
N-oxide, symphytine, symphytine N-oxide, uplandicine, uplandicine N-
oxide, echimidine, ehimidine N-oxide(Altamirano et al., 2005)
S. officinale Roots Symphytine, echimidine, isobauerenol, β-sitosterol (Tsutomu Furuya &
Hikichi, 1971)
S. officinale Roots Allantoin (E. Neagu et al., 2011 )
S. officinale Roots Allantoin (V.L. Savić et al., 2015)
S. officinale Seeds Allantoin (Al-Nimer & Wahbee,
2017)
S. officinale & S.
cordatumShoots and roots Allantoin, p-hydroxybenzoic acid, hydrocaffeic acid, rosmarinic acid,
chlorogenic acid(Dresler et al., 2017)
S. officinale Roots Rosmarinic acid, isomers of salvianolic acid A, B and C, acetyl intermedine,
acetyl lycopsamine and their N-oxides(Trifan et al., 2018)
S. officinale Roots Symphytoxide A (triterpenoid saponin) (Ahmad et al., 1993)
S. officinale Callus, leaves,
leaf-stalks,
ovaries, anthers
and rootsFructan (Abou-Mandour et al.,
1987; Haaß, Abou-
Mandour, Blaschek, Franz,
& Czygan, 1991 )
S. officinale Roots Bidesmosidic hederagenin hexasaccharide (Mohammad et al., 1995 )
S. officinale Seeds Γ-linolenic acid (Yunusova et al., 2017)
S. officinale Herb and roots Caffeic, p-coumaric and m-hydroxybenzoic acids (Grabias & Swiatek, 1998)
S. tuberosum Whole plant Anadoline, echimidine (Ulubelen & Öcal, 1977)
S. x uplandicum Nyman
(syn. S. peregrinum
Ledeb.)Roots and leaves Intermedine, lycopsamine, 7-acetyl intermedine, 7-acetyl lycopsamine,
uplandicine, symlandine, symviridine, myoscorpine, symphytine, echimidine(Culvenor et al., 1980 ; E.
Roeder, 1995)

62Table 2. Content (ng/g) of pyrrolizidine alkaloids from different parts of Symphytum officinale L.
(Avula et al., 2015).
Plant part Compounds Amounts (ng/g)
Roots Intermidine 280–12400
Roots Lycopsamine 800–15000
Stems Intermidine 8220
Stems Lycopsamine 1000
Leaves Intermidine 160–6600
Leaves Lycopsamine 180–3400

63Table 3. Bioactivities of different Symphytum species.
Species Plant part Bioactive compounds Bioactivity Effect Reference
S. officinalis Roots Phenolic compounds Antioxidant Higher in vitro antioxidant activity than ascorbic
acid(E. Neagu et al., 2011 )
S. officinalis Roots Phenolic acids Antioxidant High antioxidant activity of the extract Sowa et al., 2018
S. officinalis Roots Poly[3-(3,4-
dihydroxyphenyl)glyceric acid]Antioxidant Interfere in the formation of active oxygen
species(V Barbakadze et al.,
2009)
S. officinalis Roots Phenolic compounds Antioxidant Scavenge free radicals, reducing 15-LO
inhibition(Trifan et al., 2018)
S. officinalis Leaves Phenolic compounds Antifungal Inhibit the germination of fungal pathogens (Karavaev et al., 2001a)
S. sylvaticum Roots/Leaves Alkaloid echimidine-N-oxide Antifungal Strong activity against Epidermophyton
floccosum, Epidermophyton floccosum,
Nigrospora oryzae, Allefsheria boydii, Pleuretus
ostreatus, Stachbotrys atra, Curvularia lunata
and Drechslera rostrata; Moderately active
against Aspergillus niger(Kartal et al., 2001)
S. officinalis Leaves NA Antibacterial Partial and strong inhibition against mainly
Staphylococcus aureus, Bacillus subtilis,
Pseudomonas aeruginosa, Salmonella typhi(Sumathi, 2016)
S. officinalis Roots/Leaves NA Antibacterial Leaves – Inhibitory effect against Bacillus
cereus; Roots – maximum inhibitory effect
against Proteus vulgaris and Staphylococcus
aureus(Sumathi et al., 2011)
S. officinalis Roots Phenolic compounds Antibacterial Inhibition against Escherichia coli ATCC8739
and Salmonela typhimirium ATCC6538(V. L. Savić et al., 2015)
15-LO, 15-lipoxygenase; NA, Not available information

64Table 4. Summary of in vitro evidence of Symphytum plants antimicrobial effects.
Microorganism Test type Symphytum Plant Part Reference
Staphylococcus aureus, Bacillus
subtilis, Escherichia coli and
Klebsiella pneumoniaeInhibition zone diameter Aerial propylene glycol extracts (Cvetkovic, Stanojević,
Kundaković, Zlatkovic, &
Nikolić, 2015)
S. sclerotiorum Antagonistic effect by growth
inhibition zones Endophytic fungi isolated from
leaves(Rocha et al., 2009)
Staphylococcus aureus;
Pseudomonas aeruginosa;
Salmonella typhimurium; Shigella
sonnei; Klebsiella pneumoniae and
Escherichia coliInhibition zone diameter more
than 7 mmLeaves extract (Bouzada et al., 2009)
No antimicrobial activity with 34
pathogenic bacterial and fungal
isolatesDiameter of zone of inhibition Aqueous leaves extract (Woods-Panzaru et al., 2009)
Staphylococcus aureus Disc diffusion method Leaves (Borchardt et al., 2008)
Staphylococcus aureus Disc diffusion method Ethanolic plant extract (Onofre et al., 2016)
Escherichia coli, Salmonela
typhimiriumminimum inhibitory concentration
(MIC) Aqueous extract of root (Savic, Nikolic, Stanojevic, Ilic,
& Stankovic, 2012)
Bacillus cereus, Proteus vulgaris
and Staphylococcus aureusDisc diffusion method
Disc diffusion methodChloroform leaf extract
Methanol root extract(Sumathi, 2016)
Bacillus subtilis
Staphylococcus aureus, Bacillus
subtilis, Pseudomonas aeruginosa,
Salmonella typhiStreak plate method Chloroform leaf extract
Ethanol leaf extract(Sumathi, 2016)
Bipolaris oryzae Antifungal activity by Kirby-
Bauer and incubationPlant aqueous extracts (Knaak et al., 2013)
Erysiphe graminis conidia and
Puccinia graminis uredosporesWheat stem Leaves extract (Karavaev et al., 2001b)

65

66Table 5. Antioxidant effect of different Symphytum species.
Symphytum Species Extract or fraction Experimental model Key Results Reference
DPPH 0.72 µg/mL
Lipid Peroxidation 0.01 µg/mL
HR scavenging ≥ 100 µg/mLS. asperum Lepech Roots Water-soluble
hydroxycinnamate-derived
polymer
SO anion scavenging 13.4 µg/mL(Barthomeuf et al.,
2001)
DPPH 80.25 µg/mL
ABTS 20.14 µg/mL
FRAP 32.75 µg/mL S. officinale Root Ethanol extract 65%
15-LO 63.68 µg/mL (Trifan et al., 2018)
S. officinale Root Ethanolic concentrated extract DPPH 80 % inhibition (Paun et al., 2012)
S. officinale Root Hot water extraction (25 μg ) ABTS
SOD activity9.61 TE μM
48.98%(Thring, Hili, &
Naughton, 2009)
DPPH 119.96 µg/mL S. officinale L Leave Methanol extract
ABTS 193.65 µg/mL(Nossa González et al.,
2016)
DPPH 39.97 µg/mL Ethanolic extract
SO scavenging 190.76 µg/mL
DPPH 96.21 µg/mLS. officinale L Leaves
Aqueous extract
SO scavenging 307.42 µg/mL(Alkan et al., 2014)
Concentrated ethanol extract DPPH
ABTS374.67 (µmol Trolox/g)
1152.01S. officinalis roots
Concentrated methanol extract DPPH
ABTS207.81 µmol Trolox/g)
874.81 µg/mL(Elena Neagu, Roman,
et al., 2010)
S. caucasicum Leaves Ethanol extract DPPH 27.5 µg/mL (Badridze et al., 2013)
Ethanol/water extract
Concentration 100 μg/mlDPPH
FRAP0.985 TE mM/g
0.274 AAE mM/gS. officinale Root
Water extract Concentration
100 μg/mlDPPH
FRAP0.288 TE mM/g
0.065 AAE mM/g(Ireneusz Sowa et al.,
2018)
CL lum
OPZ-stimulated PMN85.4 μm/mL
125 μm/mL
CL luc
OPZ-stimulated PMN57.4 μm/mL
88.4 μm/mL S. officinale Roots Water soluble high-molecular-
weight biopolymer HP-et
(Precipitated by ethanol)
Water soluble high-molecular-
weight biopolymer
HP-ac (Precipitated by CL luc
PMA-stimulated PMN97.8 μm/mL
70 μm/mL (V Barbakadze et al.,
2009)

67acetone) CL luc in system
HX/XO1.5 μm/mL
0.7 μm/mL
CL lum
(OPZ-activated PMNs)79.6 μm/mL
113.0 μm/mL
CL luc
(OPZ-activated PMNs)82.0 μm/mL
66.5 μm/mL
CL luc
(PMA-activate d PMNs)74.6 μm/mL
107.4 μm/mLS. asperum Roots and
stemsRoots water soluble fractions
from the Stems water-soluble
fractions
Clluc
(HX/XO system)2.0 μm/mL
0.75 μm/mL(V. V. Barbakadze et
al., 2007)
CL lum
(OPZ-activated PMNs)150.5 μm/mL
149.6 μm/mL
CL luc
(OPZ-activated PMNs)108.6 μm/mL
113.0 μm/mL
CL luc
(PMA-activate d PMNs104.5 μm/mL
170.7 μm/mLS. caucasicum Roots and
stemsRoot Water-soluble fractions
Stems Water-soluble fractions
Clluc
(HX/XO system)3.2 μm/mL
3.0 μm/mL(V. V. Barbakadze et
al., 2007)
CL lum
OPZ-stimulated PMN52.0 μm/mL
CL luc
OPZ-stimulated PMN27.0 μm/mLS. asperum Leaves High molecular weight
fractions
CL luc in system
HX/XO1.2 μm/mL(VV Barbakadze et al.,
2011)
CL lum
OPZ-stimulated PMN58.0 μm/mL
CL luc
OPZ-stimulated PMN31.0 μm/mL S. caucasicum Leaves High molecular weight
fractions
CL luc in system
HX/XO1.5 μm/mL(VV Barbakadze et al.,
2011)
DPPH, 2,2‐diphenyl‐1‐picrylhydrazyl; AOS, active oxygen species; PMN, polymorphonuclear neutrophils; CL lum, luminol-induced
chemiluminescence; CL luc, lucigenin-induced chemiluminescence; OPZ, opsonized zymosan; FMA, phorbolmyristateacetate; SO, superoxide;
FRAP, ferric reducing ability of plasma; ABTS, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid); HX–XO, Hypoxanthine–xanthine oxidase;
HO, Hydroxyl radical; 15-LO, 15-Lipooxygenase; TE, Trolox equivalent; AAE, ascorbic acid equivalent.

68Table 6. Symphytum species clinical trials.
Study Design Treatment Diagnosis Patients Key Effects References
Clinical trial
randomized placebo-
controlledComfrey root-based cream
containing mistletoe or placebo
Duration: -OA knee
Adult (n=61)Greater reduction in morning/evening pain
(both 28%) and night pain (51%) in herbal
group as compared to placebo(Schmidtke-
Schrezenmeier,
1992)
Clinical trial
randomized double-
blind bi-center
placebo-controlled Comfrey root extract
(1:2, ethanol 60%, V/V, 35%)
Kytta-Salbe® f or placebo
Duration: 21 daysOA knee
Adult (n=220)Total score of pain at rest and pain on
movement improved by 54.7% in the verum
group, 10.7% in the placebo group(Grube et al.,
2007)
Clinical trial
Randomized
multicenter
reference-controlledComfrey root-based cream
containing tannic acid and
eucalyptus (4Jointz)
or reference cream with only
eucalyptus
Duration: 6 weeksOA knee
Adult (n=43)Comfrey-based creams more effective in
relieving pain, stiffness and improving
daily function than the eucalyptus reference
cream(D. B. Smith &
Jacobson,
2011)
Clinical trial
randomized bi-
center double-blind
placebo-controlledComfrey root-based cream
containing the tannic acid, aloe
vera gel, eucalyptus oil,
frankincense oil (4Jointz)
or placebo
Duration: 12 weeksOA knee
Adult (n=133)Pain scores significantly reduced in 4Jointz-
received group compared to the placebo
group(Laslett et al.,
2012)
Clinical trial
randomized double-
blind multicenter
placebo-controlledComfrey root extract (1:2,
ethanol 60%, V/V, 35%),
Kytta-Salbe® f or placebo
Duration: 5 daysBack pain
Adult (n=120)Reduction in the intensity of the pain on
active standardized movement about 95.2%
in the verum group and 37.8% in the
placebo group(Giannetti et
al., 2009)
Clinical trial
randomized
multicenter double-
blind three-arm
placebo-controlledComfrey root extract (1:2,
ethanol 60%, V/V, 35%) with
1.2% methyl nicotinate
(combination cream: Kytta-
Balsam Wf) or reference with
only 1.2% methyl nicotinate or
placebo
Duration: 5 daysBack pain
Adult (n=379)Combination cream and 1.2%methyl
nicotinate resulted in statistically significant
and clinically relevant reductions in pain
scores and increases in tenderness
compared to placebo(Pabst et al.,
2013)
Clinical trial
randomized pilot Comfrey root ointment or
placeboRheumatism
(different forms) Clearly better efficacy of comfrey ointment
as compared to placebo, regarding (Petersen et al.,
1993)

69study placebo-
controlledDuration: 4 weeks Adult (n=41) "tenderness on pressure" for patients with
epicondylitis and tendovaginitis
Clinical trial
randomized double-
blind multicenter
placebo-controlledComfrey root extract (35%),
(Kytta-Salbe® f) or placebo
Duration: 8 daysUnilateral ankle
sprains
Adult (n=142)Comfrey treatment was clinically and
significantly superior regarding the
reduction of pain and ankle edema, ankle
mobility and global efficacy(Koll et al.,
2004)
Clinical trial
randomized single-
blind multicenter
parallel-groupcomfrey root extract (35%),
(Kytta-Salbe® f) or diclofenac
Duration: 7 daysUnilateral ankle
sprains
Adult (n=164)Ankle swelling: decreased 79.5% in the
comfrey root extract group, decreased
69.4% in the diclofenac group; pain on
pressure: reduced 80.6% in the comfrey
root extract group, decreased 74.7% in the
diclofenac group.(Predel et al.,
2005)
Open uncontrolled Comfrey extract obtained from
the aerial parts of the plant
Duration: 14 daysPainful locomotor
system disorder
Adult (n=105)Very effective results in the treatment of
subacute and chronic complaints
accompanied by functional symptoms like
swellings -excellent results in 90–94 % of
patients(Kucera et al.,
2000)
Clinical trial
randomized double
blind multicenter
reference-controlled10% comfrey extract from the
aerial parts of Symphytum ×
uplandicum Nyman
(Traumaplant®) or reference
with 1% active ingredient
Duration: 14 daysAnkle distortion
Adult (n=203)Reduction of pain on active motion, pain at
rest and functional impairment found to be
highly significant, and excellent efficacy in
85.6 % of cases with verum compared to
65.7% of cases with reference(Kučera et al.,
2004)
Clinical trial
randomized
double-blind
multicenter
reference-controlled10% comfrey extract from the
aerial parts of Symphytum ×
uplandicum Nyman,
Traumaplant® or reference
with 1% active ingredient
Duration: 10 daysMyalgia
Adult (n=215)Pain on active motion, pain at rest and pain
on palpation were significantly better
reduced in verum group compared to
reference group, and no systemic adverse
effects(Kucera et al.,
2005)
Clinical trial
randomized
double-blind
reference-controlled10% comfrey extract from the
aerial parts of Symphytum ×
uplandicum Nyman,
Traumaplant® or reference
with 1% active ingredient
Duration: 10 daysFresh abrasions
Adult (n=278)Significant and clinically relevant faster
initial reduction of wound size (49 ± 19 %
versus 29 ± 13 % per day), and efficacy in
93.4 % of cases of verum group as
compared to reference group (61.7 % of
cases)(Milos Barna et
al., 2007)

70Open uncontrolled 10% comfrey extract from the
aerial parts of Symphytum ×
uplandicum Nyman,
Traumaplant®
Duration: 9 daysBlunt traumata
Children (n=196)Clear improvement in the range of 84.5 to
100% for every individual parameter such
as pain on palpitation, pain in motion,
functional impairment, edema and
hematoma(Grünwald et
al., 2010)
Clinical trial
randomized double-
blind reference-
controlled10% comfrey extract from the
aerial parts of Symphytum ×
uplandicum Nyman,
Traumaplant® or reference
with 1% active ingredient
Duration: 9 daysFresh abrasions
Children (n=108)Cream with higher active ingredient
concentration reached to 50 % healing rate
0.9 days earlier than that of the lower
concentration cream(M Barna et al.,
2012)
Pharmacological
Trial5 or 10 % of a comfrey root
extract (2:7, 50 % ethanol) or
diclofenac
Duration: -Experimentally-
induced UV-B
erythema
Adult (n=28) Greater anti-inflammatory potency of the
extract than diclofenac (or equal)(Andreas et al.,
1989)
Open uncontrolled 10% comfrey extract from the
aerial parts of Symphytum ×
uplandicum Nyman,
Traumaplant®
Duration: 4 weeksDecubitus ulcers
Adult (n=161)89.2% reduction in total decubitus area and
88% decrease in depth of the pressure ulcer(Štepán et al.,
2014)

71N
HNH
OOH
NO
H2N
NO OOO
OHHO
HO
Allantoin Echimidine
NOOO
HO
HOHH
O
NO OOO
HO
HO
Symviridine Symphytine
NOHOO
HO
HOH
NOOO
HO
HOH
O
Intermedine Acetylintermedine
NOHOO
HO
HOH
NHO OOO
OH HO
Lycopsamine Lasiocarpine
Figure 1. Chemical structure of the main heterocyclic compounds of the Symphytum plants