Tongvaren is een middelhoge, ook in de winter groene plant, met in de zomer rijpe sporen. De bladschijf (het blad) is ongedeeld, tongvormig, heeft gave randen en is glanzend lichtgroen. Sporen zitten in ‘sori’ ofwel sporenhoopjes met een vliesje, die in twee evenwijdige rijen op de onderkant van het blad te zien zijn.
Tongvaren komt voor van het Marokkaanse Atlasgebied tot bij de Kaspische Zee. Ook in Japan en Noord-Amerika. In Belgie is hij vooral massaal in de Ardennen te vinden: op doorgaans beschaduwde, vochtige plaatsen zoals oude muren, greppelkanten, bossen, onder struikgewas of op dood hout. Deze varen heeft een vochtige plek nodig om van een spoor tot een kiemplantje te ontwikkelen. Strenge vorst verdraagt hij slecht.
Latin name: Asplenium scolopendrium
Synonyms: Phyllitis scolopendrium, Scolopendrium officinarum, Scolopendrium vulgare
Family: Polypodiaceae (Polypody Fern Family)
Medicinal use of Hart's Tongue Fern: The fronds are astringent, cholagogue, diaphoretic, diuretic, expectorant, vulnerary. Externally it is used as an ointment in the treatment of piles, burns and scalds. An infusion is taken internally for the treatment of diarrhoea, dysentery, gravelly deposits of the bladder and for removing obstructions of the liver and spleen. The fronds are harvested during the summer and can be dried for later use.
Known hazards of Asplenium scolopendrium: Although we have found no reports of toxicity for this species, a number of ferns contain carcinogens so some caution is advisable. Many ferns also contain thiaminase, an enzyme that robs the body of its vitamin B complex. In small quantities this enzyme will do no harm to people eating an adequate diet that is rich in vitamin B, though large quantities can cause severe health problems. The enzyme is destroyed by heat or thorough drying, so cooking the plant will remove the thiaminase.
The Hart’s Tongue Fern is native to Europe and there is a variety of it in North America, Phyllitis scolopendrium var.americana which is smaller than the European variety. It’s a member of the spleenwort family, Aspleniaceae and grows in Asia too and parts of North Africa, and prefers moist, shady places. It can grow in woods and along river banks, as well as in walls. I have a vague memory of the leaves not having a pleasant smell when bruised, and I avoided the fern as a child because I didn’t like the waxy feel of its leaves which are shaped like the tongue of the red deer, or so it was thought, hence its name. Perhaps I didn’t appreciate it because it grew on damp walls on buildings I didn’t particularly like, such as public toilets.
It was known to the ancient Greek physician Galen (c 130-210 AD) who is deemed to be second only of the ancient Greek physicians to Hippocrates, the Father of Medicine. He used it in remedies for dysentery and diarrhoea because of its astringent qualities, in an infusion, made from 2 ounces of the leaves to 1 pint of water. This was later used by medieval physicians to remove obstructions of the spleen and liver.
Dioscorides, writing his Materia Medica in 1 AD remarked that the leaves tasted bitter, and recommended it being drunk with wine as an antidote to snake bites and for diarrhoea and dysentery.
The fronds can be harvested in summer and dried for later use. If dried, it can be made into an ointment for scalds, burns and piles. It was one of the five great capillary herbs along with the maidenhair fern which is a common house plant in often growing in pots in British bathrooms.
The mediaeval herbalists called it lingua cervina or deer’s tongue in their old herbals. Culpeper writing later, in the 17th century says “It is a good remedy for the liver” and goes on to include its benefits to the spleen and “the heat of the stomach.” He continues: -
“The distilled water is very good against the passion of the heart, to stay hiccough, to help the falling of the palate and to stay bleeding of the gums by gargling with it.”
It has been used to ease gout, clear the eyes, heal fresh wounds (juice from the leaves) reduce fevers and to get rid of warts and pistules in early European traditional medicinal systems. It is mentioned in Michael Drayton’s (1563-1631) poem, Poly-Olbion, Song XIII, referring to its use for removing stones and gravel from internal organs, “hart’s tongue for the stone.”
It has been the subject of some clinical trials which suggest that it may be effective for digestive disorders as Culpeper thought, and that it may increase production of urine as well as soften stools (as senna does) and it may stimulate the bowel to contract and empty (in which case it would be good for constipation and piles perhaps).
The physicians of Myddfai had this recipe for remaining chaste, (not involving the chaste berry), presumably for a woman rather than a man.
“If you would always be chaste, eat daily some of the herb called hart's tongue, and you will never assent to the suggestions of impurity.”
Apart from having remedies for ailments, these old physicians also gave dietary advice and here is what they had to say for the
“Month of May. Do not eat sheep's head or trotters, use warm drink. Eat twice daily of hart's tongue, fasting. Take a gentle emetic. Use cold whey. Drink of the juice of fennel and wormwood.” It isn’t clear whether this refers to the herb or the deer’s tongue, but whichever, it wouldn’t have made much of a meal; an austere diet, to be sure, but one that was perhaps followed by the adherents of the physicians of Myddfai in Wales.
Botanical: Scolopendrium vulgare; Asplenium scolopendrium (LINN.)
Family: N.O. Filices
Description
Medicinal Action and Uses
---Synonyms---Hind's Tongue. Buttonhole. Horse Tongue. God's-hair. Lingua cervina.
---Part Used---Fronds.
The Hart's Tongue, a fern of common growth in England in shady copses and on moist banks and walls, is the Lingua cervina of the old apothecaries, and its name refers to the shape of its fronds.
---Description---Its broad, long, undivided dark-green fronds distinguish it from all other native ferns, and render it a conspicuous object in the situations where it abounds, as it grows in masses. It receives its name of Scolopendrium because its fructification is supposed to resemble the feet of Scolopendra, a genus of Mydrapods. The sori are in twin oblique lines, on each side of the midrib, covered by what looks like a single indusium, but really is two, one arranged partially over the other. In the early stages of its growth, the folding over of the indusium can be clearly seen through a lens. The fronds are stalked and the root, tufted, short and stout. This fern is evergreen and easy of cultivation.
---Medicinal Action and Uses---In common with Maidenhair, this fern was formerly considered one of the five great capillary herbs.
The older physicians esteemed it a very valuable medicine, and Galen gave it in infusion for diarrhoea and dysentery, for which its astringent quality made it a useful remedy. In country districts, especially in Wales and the Highlands, an ointment is made of its fronds for burns and scalds and for piles, and it has been taken internally for Bright's Disease, in a decoction made of 2 oz. to a pint of water, in wineglassful doses. In homoeopathy, it is administered in combination with Golden Seal, for diabetes. It is specially recommended for removing obstructions from the liver and spleen, also for removing gravelly deposits in the bladder. Culpepper tells us:
'It is a good remedy for the liver, both to strengthen it when weak and ease it when afflicted.... It is commended for hardness and stoppings of the spleen and liver, and the heat of the stomach. The distilled water is very good against the passion of the heart, to stay hiccough, to help the falling of the palate and to stay bleeding of the gums by gargling with it.'
Plants (Basel). 2021 May 25;10(6):1053. doi: 10.3390/plants10061053.
Venelin Petkov 1, Tsvetelina Batsalova 1, Plamen Stoyanov 1 2, Tsvetelina Mladenova 1, Desislava Kolchakova 1, Mariana Argirova 2, Tsvetanka Raycheva 3, Balik Dzhambazov 1
Abstract
The ferns Asplenium ceterach L., Asplenium scolopendrium L. and Asplenium trichomanes L. have wide application in traditional medicine worldwide. However, the scientific research on their anticancer and antibacterial properties is insufficient. The present article aims to provide more information on this topic. Extracts derived from the aerial parts of A. ceterach, A. scolopendrium and A. trichomanes were examined using a panel of in vitro assays with different bacterial and mammalian cells. The cytotoxicity and anticancer activity of the samples were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays with three human (A549, FL, HeLa) and three murine (3T3, TIB-71, LS48) cell lines. Inhibitory effects on the growth of Gram-positive (Bacillus cereus) and Gram-negative (Pseudomonas aeruginosa) bacteria were determined by the agar diffusion assay. Apoptosis-inducing properties of the extracts were analyzed by flow cytometry. Superoxide dismutase (SOD) activity in extract-treated cells was investigated by ELISA. The obtained results demonstrate selective anticancer activity of all three Asplenium species. The extract from A. ceterach displayed the strongest inhibitory properties against human cervical cancer cells and bacterial cells. It induced a lower level of cytotoxicity against mouse cell lines, indicating a species-specific effect. The extract from A. trichomanes demonstrated better anticancer and antibacterial properties than the sample from A. scolopendrium. Further experiments linked the mechanism of action of A. ceterach extract with oxidative stress-inducing potential and strong proapoptotic potential against the cervical cancer cell line HeLa. A. trichomanes and A. scolopendrium extracts appeared to be potent inducers of necrotic cell death.
The scientific name of this genus and its common name (“spleenwort”) stem from an old doctrine that Asplenium plants are useful for treatment of spleen diseases due to their spleen-shaped spores. Indeed, many of these ferns have been used for centuries in traditional medicine in different countries worldwide but their application significantly exceeds therapy of spleen disorders [1,2]. It has been documented that nine Asplenium species grow in Bulgaria [3]. Among them are A. ceterach, A. trichomanes and A. scolopendrium—well characterized and widely distributed ferns in Europe. Numerous applications in folk medicine have been reported for these three species. A. ceterach has been used for relief of spleen and kidney stones complaints, hemorrhoids, hypertension, respiratory and intestinal disorders, as well as a diuretic, expectorant, laxative, etc. [1,4,5]. A. scolopendrium has been used for the treatment of wounds, bleeding, myalgia, lung, liver and spleen diseases, inflammation of gums, urogenital system disorders, and as a mild laxative, diuretic, astringent, diaphoretic and tonic [6,7]. A. trichomanes has been applied to relieve cough and renal conditions, as a laxative, expectorant, abortifacient and emmenagogue [8,9]. Its estrogenic activity in vitro has been proven [8], explaining its administration as an emmenagogue.
Plant-derived extracts often represent a rich source of natural compounds with beneficial medical effects. The importance of such substances has been proven since ancient times [10]. Nowadays, purified compounds of plant origins offer a promising alternative to synthetic drugs and have made a major contribution to the therapy of cancer and infectious diseases, but also to the therapy of cardiovascular and autoimmune diseases [10,11]. Their application in drug discovery and development was initiated in the 19th century [10]. Since then, this vast research area continues to expand and present new perspectives for improved pharmaceutical products and development of new drugs with potential to combat incurable diseases [12]. Hence, the present study aims to contribute to this field by investigating the biological activity of three Asplenium species growing in Bulgaria—A. ceterach, A. trichomanes and A. scolopendrium. Research on their cytotoxicity against different cell types, as well as anticancer and antibacterial potential, is insufficient. Therefore, the present work aims to unravel this scientific question. Aerial parts from A. ceterach, A. trichomanes and A. scolopendrium growing in Bulgaria were collected and used for preparation of extracts. The obtained samples were analyzed by different in vitro assays with mammalian and bacterial cells. The extracts demonstrated a selective inhibitory effect against a cervical cancer cell line and low cytotoxicity against noncancerous human and mouse cells. A. ceterach and A. trichomanes extracts displayed antibacterial activity.
References
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Interest in the pharmacologic role of bioactive compounds present in plants has increased in the last decade. One of the most investigated class of natural compounds are the so-called “phytoestrogens” a family which embody several groups of non-steroidal compounds including isoflavones, lignans and coumarins. Epidemiologic data indicate that the Asian people have lower rates of osteoporotic fractures, cardiovascular diseases, postmenopausal symptoms and certain cancer than the western population. These health advantages are notably reduced when Asians adopt a western lifestyle diet. Genistein and daidzein are present in the soy which is very important food for Asian population. Thus many researcher are investigating the relationship between, for example, soy intake and postmenopausal disease (Usui, 2006, and references therein).
The two known forms of estrogen receptors, namely ERα and ERβ, are differently distributed in tissues and certain phytoestrogens, for example genistein or apigenin, display ERβ binding selectivity (Miller et al., 2003) while the 17β-estradiol displays similar affinity for both the receptors. Thus, the investigations on natural compounds with estrogenic effects could be the source for the development of new classes of SERMs (selective estrogen receptors modulators). Recently SERMs have become available for the treatment of osteoporosis and there are clinical studies being performed to examine their effects on cardiovascular disease and breast cancer (Usui, 2006, and references therein). Nowadays there is the need for compounds, which shows estrogenic activity for example on bone with little effects on uterus or breast tissues; thus the search for improved estrogen like compounds with alternate profile has continued.
In our research on phytoestrogen compounds we started from plants which are used, in the traditional medicine, for the treatment of menstrual pain, as contraceptives, or as menopausal remedies, etc. (Innocenti et al., 2007). In this paper, we consider Asplenium trichomanes L. (Aspleniaceae) a fern widely distributed in the Italian temperate regions. Few phytochemical data are present in the literature about this plant three kaempferol derivatives have been reported from Asplenium trichomanes L. by Imperato (1979). More literature data are available for different Asplenium species. Several flavonoid glycosides (Iwashina et al., 1990, Iwashina et al., 1993, Iwashina et al., 1995, Iwashina et al., 2000, and references therein; Mizuno et al., 1990, Mizuno et al., 1991, Imperato, 1992, Imperato, 1993, Umikalsom et al., 1994) and xanthone glycosides (Imperato, 1991, and references therein) have been isolated from Asplenium species. Asplenium bulbiferum is rich in flavonoids with antioxidant proprieties and this species is used for nutritive properties by Maori people (Cambie and Ferguson, 2003). Asplenium nidus was used in the traditional Vanuatu medicine as contraceptive as well as to reverse sterility (Bourdy et al., 1996, and references thereby).
In the Italian folk medicine Asplenium trichomanes was used as an expectorant, anti-cough remedy and laxative (Pomini, 1990) as well as emmenagogue (Negri, 1979). In North America the infusion of this species was used as abortifacient and for irregular menses (Moerman, 1998). The aim of our study was to assess the in vitro estrogenic properties of tea, decoction and crude extract of Asplenium trichomanes using the MCF7 and SK-NBE cells test. In addition phytochemical investigations were carried out yielding in the isolation of two new glycoside phenol derivatives and six known compounds which were also preliminary evaluated for their estrogenic effects with the same tests.
Asplenium trichomanes was used as an expectorant, anti-cough remedy, laxative, emmenagogue, abortifacient and for irregular menses.
Aim of the study
To investigate the in vitro estrogenic activity of Asplenium trichomanes extracts and isolated compounds and their ability to activate ERα and ERβ.
Materials and methods
Leaves infusion (IF), decoction (DC) and methanol extract (ME) were prepared. MCF7/EREluc cell line which expresses endogenous ERα, and SK-NBE cells transiently transfected with the estrogen receptors (ERα and ERβ) were used for the estrogenic activity assays. Phytochemical investigations were performed (CC, HPLC, etc.) and structure of isolated compounds were achieved on the basis of 1D and 2D NMR techniques and HR-MS spectrometry.
Results
IF and ME were active in our MCF7 model; selectivity for the ERβ receptor was observed in the SK-NBE test. Two new phenol derivatives, 4-vinyl-phenol-1-O-[α-l-rhamno(1 → 6)-β-d-glucopyranosyde] (1) and kaempferol-3-O-α-[2′acetyl]-arabinofuranosyl-7-O-α-l-rhamnopyranoside (2) were isolated with six known compounds (3–8). Compounds 2–4, 7 and 8 showed selectivity for the activation of the ERβ receptor although with a moderate activity compared with 17-β-estradiol.
Conclusion
Further investigations about the estrogenic effects of this plant are needed but our data can, at least in part, explain some of its traditional use as emmeagogue.
Although ferns have been poorly studied from a phytochemical and nutritional approach, fronds are the main part of the ferns used to make medicinal preparations by the Malayalis in Kolli hills, India (9). The election of the extraction solvent is a relevant aspect in the phytochemical determination of plant drugs. Selectivity (based on polarity target phytochemical), safety, neutral, and easy to separate from the rest of no target phytochemicals, low viscosity, low boiling temperature, and economical are the criteria for selecting the extraction solvent (47). Currently, most plant matrices are obtained from organic solvents compared to other solvents such as green solvents (environmentally friendly). For the in vitro studies that we have carried out on A. trichomanes and C. officinarum extracts, we have used the methanolic extract of their fronds while the correspondence hexane extract was discarded due to the lower presence of total polyphenol content (TPC) than the corresponding methanolic extract in a previous study (34).
In the Russian Far East, there are a minimum of 13 fronds of edible fern species, where P. aquilinum predominates (11). The degree of maturation of the fronds (young fronds, as known fern shots or fiddleheads, or mature fronds) and the way in which the fronds are preserved (by freezing, canning, salting, or drying) are important to determine the potential toxicity of edible ferns. For example, different government dossiers regard the safeguard of public health against P. aquilinum to whose toxins affect human populations and animals. The main toxin that triggers the carcinogenesis of P. aquilinum is ptaquiloside (an illudane, a subclass of sesquiterpenes) (48, 49). Currently, ferns are not incorporated into the diet of European countries. However, the publication of nutritional properties of plant matrices of ferns may be an incentive to incorporate ferns in European diets (14).
We have considered the methanolic extracts of the fronds of A. trichomanes and C. officinarum as potential reservoirs of phytochemicals. Derived that other Aspleniaceae species have been described as reservoirs of phytochemicals. Such as the ferns Asplenium adiantum-nigrum L. (Aspleniaceae) and Asplenium ruta-muraria L. (Aspleniaceae) (50), of which the methanolic extract of the fronds of both ferns is the part with the highest total phenol content (TPC) and total flavonoid content (TFC). And specifically, the content of flavonoids in the fronds is relevant due to one of the functions of the flavonoids in the fronds, which are the neutralization of UV radiation and ROS (51). In the phytocharacterization by HPLC-MS/MS of ATM fronds, a greater quantity of flavonoids, especially flavonols related to kaempferol, was obtained, as Dall’Acqua et al. (52) described. In our phytocharacterization, the main phytochemical isolated from ATM has been hyperoside, another flavonol. We have also isolated kaempferol and kaempferol glycoside (kaempferol-3-glucoside). Resulting that flavonols are the main polyphenolic species determined in ATM, representing 68.8% of the total phytochemicals determined by HPLC-MS/MS. In the case of COM, flavonoids are minor phytochemicals detected by HPLC-MS/MS compared to phenolic acids. These results are equivalent to that obtained by Zivkovic et al. (53). In contrast to Zivkovic et al. (53), we have detected fractions of rutin in the fronds of C. officinarum. In this study, chlorogenic acid is the main phytochemical determined in COM, while the other cinnamic acids such as caffeic acid are only detected in traces. This observation has already been previously reported by Tomou et al. (28). Phytocharacterization by HPLC-MS/MS of both extracts coincides with the nature of the phytochemicals determined by TLC in a previous study (34).
Durdevic et al. (54) determined that the content of phenolic species in the ethylacetate extracts of A. trichomanes and C. officinarum is higher in the frond than in the correspondence rhizome. And the predominance of aglycone phytochemicals (no sugar phytochemicals) than glycone phytochemicals (sugar phytochemicals) in the ethylacetate frond extract. We have determined a higher amount of TPC in the methanolic extract of the fronds of C. officinarum than in the methanolic extract of the fronds of A. trichomanes, as described by Durdevic et al. (54).
Currently, there is a great discrepancy in the cytotoxicity of ferns, derived from the fact that they are a widely diversified taxon. However, in the cytotoxic determination of methanolic extracts, different species of ferns have concluded greater cytotoxicity in the frond than the corresponding rhizome (55). For this reason, it is important that cytotoxicity assays have been performed in a wide range of concentrations. The most common assays to determine cell viability are the reduction in the tetrazole salt or MTT, the uptake of the Neutral Red dye (NRU), or the release of lactate dehydrogenase among others (42). The measurements of most of these techniques are end point and present advantages and disadvantages. In this sense, MTT is usually the method of choice in different cytotoxic studies (38), because it has proven to be valid with different cell lines and is relatively straightforward and useful when conditions are optimized. Moreover, the MTT assay has been used to characterize the cytotoxic profile of different plant extracts including ferns (34). Actually, MTT is regarded as a gold standard of initial cytotoxicity assays as it is highly sensitive and a high-throughput screening assay together with its low economic cost (56). Nevertheless, the NRU test failed to be sensitive in our case. We are also aware that in our research there may be potential interferences of the extracts in the assays by colorimetric methods. It is described that some phytochemicals can interact with MTT producing false-positive cell viabilities (57). For this reason, the absence of interference has been verified by the MTT assay for the in vitro concentrations of the extracts tested. Petkov et al. 2021 (58) analyzed the cytotoxicity of methanolic fronds extracts of three Aspleniaceae ferns by MTT, of which two were A. trichomanes and C. officinarum. The comparison of our cytotoxicity results with the results of Petkov et al. 2021 (58) is relevant due to the same methodology for obtaining the frond extract (frond methanolic extract) and for determination of cell viability (MTT assay) for A. trichomanes and C. officinarum. In the initial toxicity studies of the pharmaceutical industry, assays with a high sensitivity against cytotoxicity with reasonable cost are used, in which various hepatic cell lines are generally used, such as HepG2 (59). The absence of cytotoxicity of the extracts in HepG2 confirms the safety of these extracts.
A diet rich in antioxidants and polyphenols contributes to reducing the risk of diseases resulting from oxidative damage. However, the employment of synthetic antioxidants, as in the case of butylated hydroquinone, in recent years, has been decreased for safety reasons (51). For this reason, investigations of new plant matrices with a high content of antioxidants have increased (60). Despite the low number of studies on ferns, the methanolic extract of their fronds is considered plant parts with a high amount of antioxidant phytochemicals (61, 62). Currently, the extracts of Polypodiaceae ferns such as Polypodium leucotomos (aqueous extract) and Polypodium vulgare L. (methanolic extract) have been reported as antioxidant and cytoprotective agents in vitro (63, 64). However, few ferns of the Aspleniaceae family, despite being the main ferns in Europe, have been studied as plant matrices for the contribution of antioxidant phytochemicals (14). For example, in the methanolic extract of A. adiantum-nigrum and C. officinarum (both species are Aspleniaceae family), mangiferins and mangiferin-related phytochemicals have also been isolated among other polyphenolic phytochemicals (50, 53). This fact demonstrates the potential of the Aspleniaceae as a reservoir of polyphenolic phytochemicals.
Conclusion
The methanolic extracts of the fronds of the two main species of ferns from the Prades mountains, A. trichomanes and C. officinarum, present interesting phytochemicals of different nature without being cytotoxic in the mouse fibroblast 3T3, human keratinocyte HaCaT, cervical human cancer HeLa, liver human cancer HepG2, breast human cancer MCF-7, and lung human cancer A549 cell lines at the assayed concentrations. This fact provides new evidence for considering certain ferns of the Aspleniaceae family as plant matrices for the extraction of phytochemicals of pharmaceutical or nutritional interest.