Ethnobotanical Survey of Dracaena cinnabari and Investigation of the Pharmacognostical Properties, Antifungal and Antioxidant Activity of Its Resin

Dracaena cinnabari Balf. f. (Dracaenaceae) is an important plant endemic to Soqotra Island, Yemen. Dragon’s blood (Dam Alakhwin) is the resin that exudes from the plant stem. The ethnobotanical survey was carried out by semi-structured questionnaires and open interviews to document the ethnobotanical data of the plant. According to the collected ethnobotanical data, the resin of D. cinnabari is widely used in the traditional folk medicine in Soqotra for treatment of dermal, dental, eye and gastrointestinal diseases in humans. The resin samples found on the local Yemeni markets were partly or totally substituted by different adulterants. Organoleptic properties, solubility and extractive value were demonstrated as preliminary methods to identify the authentic pure Soqotri resin as well as the adulterants. In addition, the resin extracts and its solution in methanol were investigated for their in vitro antifungal activities against six human pathogenic fungal strains by the agar diffusion method, for antioxidant activities using the DPPH assay and for cytotoxic activity using the neutral red uptake assay. The crude authentic resin dissolves completely in methanol. In comparison with different resin extracts, the methanolic solution of the whole resin showed the strongest biological activities. It showed strong antifungal activity, especially against Microsporum gypseum and Trichophyton mentagrophytes besides antioxidant activities and toxicity against FL-cells. These findings confirm and explain the traditional uses of the resin for the treatment of skin diseases and mouth fungal infections.


Introduction
Dragon's blood is the English common name which has been applied to the red coloured resins that are obtained from six different plant genera, Dracaena, Daemonorops, Calamus, Pterocarpus, Eucalyptus and Croton [1][2][3]. Besides Dracaena cinnabari, the genus Dracaena is represented by some other species, e.g., D. ombet and D. serrulata that are distributed in tropical Africa and southwest Arabia and D. draco, D. tamaranae from the Canary Islands and Morocco [4]. D. cinnabari Balf. f. (Dracaenaceae) is endemic to Soqotra Island, Yemen (Figure 1), in which more than 308 plant species have been considered to be endemic [4,5]. The resin of D. cinnabari from Soqotra is well known as a traditional remedy with a long history [4]; it has been traded as medicine and as a colourant agent for use in works of art for centuries, e.g., it has been discovered in some art works in a museum in Germany [3]. The original source of dragon's blood resin is believed to be D. cinnabari from Soqotra [1] (Figure 1) but numerous different resins produced from different plant species are available on the international markets under the name of Dragon's blood [3]. Therefore, some methods have been studied to differentiate these species red resins, e.g., solubility, gas chromatography coupled to mass spectrometry [3], Raman spectroscopy [1,2] spectrometry [3], Raman spectroscopy [1,2] and UV spectroscopy. Flavylium chromophores (7,4′dihydroxyflavylium) [6], have been identified to be responsible for the red colour of D. cinnabari resin, while other flavylium derivatives are responsible for the red color of the other species resins that are also called Dragon's blood [6]. The Soqotri resin is the most famous crude drug used in the traditional medicine in Soqotra Island [4,5,7]. Moreover, it is imported from Soqotra in order to be used in other traditional medicine systems all over the world, especially for the treatment of skin diseases [2,4], beside other Dragon's blood resins that have been reported in different traditional medicine systems in the world [4,8].
In the course of our biological and phytochemical studies of medicinal plants from Yemen [5,7,16], we investigated the resin of D. cinnabari. Different ethnobotanical data about D. cinnabari have been reported by Al-Fatimi [5] and by Miller [4]. Those are compared with our ethnobotanical survey in the present study. The aim of this study was to document the ethnobotanical data and the traditional medicinal and non-medicinal uses of the plant and its resin. The pharmacognostical and organoleptic properties of the D. cinnabari resin were determined for the first time in this study. These findings can be used for the identification and quality control of the resin in case of adulterations. In addition, the in vitro antifungal activity of the resin against some human pathogenic fungal strains and its antioxidant activity have been investigated to confirm the traditional medicinal uses.

Botanical Data
Dracaena cinnabari Balf. f. (Dracaenaceae) is the botanical name of an endemic wild tree, growing on Soqotra Island (Figure 1). Dragon's blood is the English common name for both tree and resin. The Arabic name "Dam Alakhwin" means "Brother's blood" and is also used for both tree and its resin ( Figure 1, Table 1). In contrast, there are two different Soqotri names for the tree and its resin. The local Soqotri name of the endemic plant (tree) is "Ahrrebo", while the Soqotri name of the authentic pure superior resin is "Emzoloh" (Figure 1, Table 1). All informants (100%) reported the two Soqotri local names of the plant and the resin. Only 40% held knowledge about the Arabic name "Dam Alakhwin". Nearly all informants (95%) reported that the authentic resin with the best quality "Emzoloh" exudes from the stem bark of the female tree of D. cinnabari, while 5% reported both male and female trees as source for the resin.

Botanical Data
Dracaena cinnabari Balf. f. (Dracaenaceae) is the botanical name of an endemic wild tree, growing on Soqotra Island (Figure 1). Dragon's blood is the English common name for both tree and resin. The Arabic name "Dam Alakhwin" means "Brother's blood" and is also used for both tree and its resin ( Figure 1, Table 1). In contrast, there are two different Soqotri names for the tree and its resin. The local Soqotri name of the endemic plant (tree) is "Ahrrebo", while the Soqotri name of the authentic pure superior resin is "Emzoloh" (Figure 1, Table 1). All informants (100%) reported the two Soqotri local names of the plant and the resin. Only 40% held knowledge about the Arabic name "Dam Alakhwin". Nearly all informants (95%) reported that the authentic resin with the best quality "Emzoloh" exudes from the stem bark of the female tree of D. cinnabari, while 5% reported both male and female trees as source for the resin. The different ethnobotanical uses of D. cinnabari resin in the study area can be classified into two main groups: medicinal and non-medicinal uses ( Table 2). All informants reported the two main groups of the plant resin uses. A.
Non-medicinal uses: The non-medicinal uses of the resin were divided into two categories-cosmetic and dye uses-according to ethnobotanical data. 95% of the informants reported the use of resin by women as lipstick and nail varnish and as smoothing and softening agent for women faces. All informants reported the use of the red resin as colourant agent for bottles and pottery vessels. No informant reported the use of the plant as food source for humans but 10% of the informants reported its fruit as food for livestock ( Table 2). B.
Medicinal uses: The field survey on D. cinnabari was conducted to provide information about established applications of the plant in the traditional medicine. 85% of the informants rely on traditional uses of the endemic medicinal plants of the island. All the local informants (100%) reported medicinal uses of the plant resin in Soqotra. The survey indicated wide uses of the resin against different ailments. Twenty diseases were reported to be treated using D. cinnabari. These ailments were classified into five categories as summarized in Table 2. Dermal diseases and dental ailments are most frequently treated with the resin (100% responses, for each), followed by eye diseases (98%), internal and external bleeding (95%) and gastrointestinal diseases (94%) ( Table 2). Treatment of wounds and dental diseases are reported in the present study for the first time ( Table 2). The resin of the endemic D. cinnabari is used extensively in Soqotra and also in inland Yemen as powerful medicinal herb [4,5]. This survey confirmed some medicinal uses that have been already reported, such as treatment of skin diseases, stomach pain, bleeding and inflamed eye [4,5,7]. Among the informants, knowledge about toxic effects of the resin is very limited. 80% of the informants reported an abortive effect in pregnant women.

Used Plant Part
All informants (100%) reported the resin as dominant part used of the plant; only 5% of them reported the leaves as additional part used ( Table 2).

Modes of Preparation
The preparations (traditional forms) are made from dried (50% of preparation modes) or fresh resin (50%): (A) Dried resin can be used as powder (40% of preparation modes) or as suspension (33%). The dried resin is crushed to powder or suspended in little water. (B) Fresh resin is crushed with little water to prepare a paste (20%). For internal use, fresh resin is chewed or its powder is taken ( Table 2). Powdered resin may be mixed with curcuma or antimony for external use while it is suspended in milk for internal use ( Table 2). The usual preparation mode is powdered dried resin.

Quantitative Analysis of Ethnobotanical Data
Ethnobotanical knowledge about D. cinnabari is very similar among the local people in Soqotra Island, therefore similar reports were collected from all informants. The Use Value (UV) demonstrates the relative importance of D. cinnabari among indigenous people in Soqotra Island. According to the quantitative analysis, the use value index of D. cinnabari is high (1.95) ( Table 3).
The Fidelity Level (FL) is calculated to indicate the efficiency of D. cinnabari to cure a given ailment; it assesses the informants' consensus about the therapeutic value of a plant species [17]. According to the calculated FL levels that range between 55-100% (Table 3), D. cinnabari is identified as an important medicinal plant species in the traditional medicine in Soqotra to treat various ailments. The endemic plant showed highest FL levels for the treatment of skin and head sores (FL = 100%), followed by conjunctivitis (95%), tooth decay (94%), bleeding (postpartum haemorrhage) 85% and stomach pain (55%) ( Table 3). These data may serve as a guide for further biological investigations [18] in order to isolate bioactive compounds.

Soqotri Standard Resin
The Soqotri resin (Dragon's blood = Dam Alakhwin) is the pure red blood resin with high quality that is known on the Island under the Soqotri name "Emzoloh". It is collected from the incision of the young stem bark of the female tree; we can describe this standard pure resin as an authentic superior Soqotri resin. The organoleptic and some physical properties of the resin were determined in this study. Like other important crude drugs, the Soqotri authentic resin may be adulterated [19]. Samples were found on the local markets in Soqotra and in inland Yemen that were adulterated by different substitutes (adulterants). Those can be classified into two main adulteration types: adulteration by partial substitution and/or by total substitution.

Adulteration by Partial Substitution
Three partial adulterations types were found in this survey. We can describe these samples according to types of adulteration for crude drugs described in pharmacopoeias [19].

•
A sample was a dark resin found on the markets in Soqotra Island and Aden with low quality. This resin type was mixed with bark pieces and sold as second value resin; in case of very low production of the pure resin (Table 4). We can describe this resin product as inferior resin.

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If the resin is mixed intentionally with bark pieces to sell as pure resin "Emzoloh", in this case it is described as a sophistication adulteration type (Table 4).

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Sometimes the resin is adulterated by accident or careless addition of small pieces of stem barks; this product can be described as an admixture adulteration.

Adulteration by Total Substitution
On the other hand, two samples were found in the markets of Aden and Sana'a city which both were totally substituted by red adulterants, which were non-resinous products; each was available in the markets under the name "Dam Alakhwin" ( Table 4).
The red coloured powder used as adulterant may have two natural origins: a plant root from Soqotra and a red coral from Indian Ocean (Table 4). Both adulterants were found on the markets of crude drugs in the inland but not on Soqotra Island. Both were sold as red power under the name "Dam Alakhwin" (Dragon's blood)

•
Euclea divinorum Hiern (Ebenaceae) grows on Soqotra Island. The plant is locally named "Kala"; its dried roots are used traditionally in Soqotra as toothbrush or as red powder for cleaning teeth and as mouthwash antiseptic agent [7]. However, the plant root and/or its red powder is imported from the Island to the inland of Yemen including Aden City markets, where the dried root pieces and the red powder were available under the name of "Dam Alakhwin" (Table 4).

•
A red powder of a red coral from Indian Ocean was found on the market in Sana'a City, called "Dam Alakhwin" and used for tooth cleaning ( Table 4).
The total substitution by adulterants like these is known as substitution adulteration in the pharmacopoeias [20] (Table 4).

•
The authentic Soqotri resin may be substituted totally by other red resins obtained from other plant species that each have the same name "Dragon's blood" and are found on the international markets.
Therefore, further morphological, organoleptic, chemical, physio-chemical and spectroscopical methods must be studied to identify and authenticate the Soqotri authentic pure resin and the adulterants. The pharmacopoeias contain a paragraph for important official crude drugs that involve the usually known adulteration types that they may be found on the international markets [19]. The documentation of common adulteration types can help to identify the authentic pure Soqotri resin either for the medicinal use or for research purposes.

Organoleptic Properties of the Resin
The Soqotri resin is an exudate product obtained from the stem of female D. cinnabari tree. The suitable time of collection is from February to March. This valuable pure resin with high quality can be described as superior authentic Soqotri resin. The dried resin is a hard, transparent brittle crystalizable exudate in lump or tear like form. Its colour is glossy deep red to purple (Figure 1). There is a second resin product with low quality that has a lump form containing bark pieces in the resin. This inferior resin is a dark red and hard amorphous material (Table 4). Dried pure resin is very weakly volatile and without characteristic smell, taste is weakly mucilage. These findings are due to the neutral character of the resin. Its suspension in water shows pH value 5.9. The organoleptic properties are an important primary examination for the quality control of crude drugs [20], therefore these results can be used in the identification and authentication of the Soqotri original superior resin.

Extractive Value of the Resin
The determination of the alcohol-soluble and water soluble extractive values is used for quality control of crude drugs [19,20]. Five solvents with increasing polarity were used for determination of the extractive values of two resin types of D. cinnabari (superior pure resin and inferior resin) ( Table 5).
Inferior resin, which is mixed with barks showed different extractive values compared to standard results of the pure superior authentic resin (Table 5). Interestingly, methanol and water extractive values showed clear differentiation between pure and inferior resins. The methanol extractive values were 100% and 90% for pure superior resin and inferior resin, respectively. Water showed no extractive result for superior resin but showed a 10% extractive value for inferior resins. Superior pure resin is dissolved completely in methanol while it is not able to be dissolved in water (Table 5).

Solubility of the Resin
The solubility of the two resin types in different solvents is given by descriptive terms according to the parts of solvent required for one part resin [19] ( Table 5). The pure authentic resin of D. cinnabari is completely soluble in methanol (one part of solvent required for one part resin), this equals to the descriptive term "easily soluble" in methanol but it is "insoluble" in water and n-hexane. In contrast to the pure resin, the inferior resin is not completely soluble in methanol, due to its bark pieces content. According to the required parts of methanol, it is "freely soluble" in methanol while it is "slightly soluble" in water and "very slightly soluble" in n-hexane ( Table 5).
The first method to differ and identify these resins by solubility tests using non-polar solvents such as chloroform has been reported 1883 [20]. Historically, various resins known as Dragon's blood differ widely from one another in their purity and appearance [21]. Generally, resins are known to be dissolved in alcohol but insoluble in water [20], which is consistent with present results of the solubility values of the Soqotri pure resin (Table 5). Therefore, the methanol and water solubility and extractive values can be used as a preliminary test and as important marker to show if the crude drug is a resin, non-resin or adulterated resin (adulterant). If the results are standard, this allows further specific examinations such as spectroscopical methods to identify the resin and its plant species. Moreover, both solubility and extractive values can be employed also for (a) the evaluation the purity of the authentic resin of D. cinnabari and, for (b) identification of the adulteration of the pure resin.

Antifungal Activity of the Resin
The FL value of D. cinnabari resin to treat skin diseases was the highest value in the survey (Table 3). Therefore, the antifungal activities of the pure resin and its extracts have been studied against six human pathogenic fungi for the first time (Table 6). This study analyzed the antifungal activity of D. cinnabari resin by measuring the growth inhibition zone in a paper disc diffusion assay ( Table 6). The best antifungal activity was demonstrated by the methanolic solution of the crude resin (20-30 mm) against A. fumigatus, M. gypseum and T. mentagrophytes followed by less polar dichloromethane and ethyl acetate extracts (18 to 20 mm), compared with the antifungal reference nystatin. In contrast, the residue of the resin dissolved in methanol showed weak activity ( Table 6). The methanolic solution of the whole resin was evaluated for the MIC. The resin solution showed a MIC at 500 µg/mL with inhibition zones 8 mm and 10 mm towards M. gypseum and T. mentagrophytes respectively.
Compared to antibacterial activities determined previously [22][23][24], the human pathogenic fungi tested appeared to be more susceptible to the resin extracts than the bacterial stains. Previous studies have reported the antifungal activities of D. cinnabari resin against three other species Candida albicans, Aspergillus flavus, and A. niger [22][23][24]. Polyphenols including flavonoids, are the main constituents of the D. cinnabari resin [9,10,14] as well as resins obtained from other species of Dracaena genus such as Dracaena cochinchinensis [8,25], Dracaena draco and Dracaena tamaranae [26]. Phenolic compounds are known to exhibit antimicrobial activities [20]. Red resin of Dracaena cochinchinensis from China has been reported to have antifungal activity [8]. Some other red resins also showed specific antifungal activities such as the resin obtained from Croton urucurana, which is called as Dragon's blood, too [27]. These findings suggest that the Soqotri resin has specific stronger antifungal but lesser antibacterial activity. Table 6. Antifungal activity of the extracts of the pure resin of D. cinnabari in an agar diffusion test.

Microorganisms
Diameter

Antioxidant Activity
The results of antioxidant assay compared with the effect of ascorbic acid as positive control are shown in Table 7. The methanolic solution of the whole pure resin showed strong antioxidant activity (IC 50 = 25.5 µg/mL), while the ethyl acetate extract showed considerable antioxidant activity (IC 50 = 40.5 µg/mL) and the residual methanolic fraction showed weak antioxidant activities (IC 50 = 153.5 µg/mL), compared to ascorbic acid (15.5 µg/mL) ( Table 7). These results indicated that phenolic compounds with low polarity in the ethyl acetate extract are responsible for the main antioxidant activity of the resin, which can be potentiated synergistically by resin compounds contained in the methanolic solution. Polyphenols are known as antioxidant natural products and have also been reported as constituents of D. cinnabari resin [9,10,14] and identified as chemotaxonomic marker for species of Dracaena genus [8,25,26]. Nevertheless, the antioxidant activity of the methanolic solution of the whole resin was reported in the present study for the first time. Interestingly, the antioxidant activity of the solution of the whole resin dissolved in methanol was stronger than all other polar and non-polar extracts that are reported in this study. Table 7. Cytotoxic activity against FL cells and free radical scavenging activity in the DPPH assay for the solution and extracts of D. cinnabari pure resin.

Free Radical Scavenging Activity Cytotoxicity against FL-Cells
The strong antifungal and antioxidant activities of the whole raw resin (as solution in methanol) confirm the traditional preparation using the whole crude resin as powder in Soqotra.

Cytotoxic Activity
The results of the cytotoxic activity against the FL-cells, a human amniotic epithel cell line, are shown in Table 7. The resin solution showed low cytotoxicity (IC 50 = 300.0 µg/mL) against FL-cells measured by the neutral red uptake assay. Extracts showed less cytotoxic activity than the whole resin (IC 50 > 500.0 µg/mL). These findings confirm the safe uses of the Soqotri resin traditional medicine. The informants in the ethnobotanical survey reported no toxic effects concerning the oral use except by pregnant women where it may cause abortion [5].

Conclusions
D. cinnabari is a famous wild endemic plant from Soqotra Island, where all local people use it for various ailments. These results provide pharmacognostical properties to identify and authenticate the Soqotri superior resin and its possible adulterants. Moreover, the ethnobotanical data of the resin led us to investigate its antioxidant and antifungal activities. The solution of Soqotri pure resin in an alcoholic solvent showed the strongest antifungal and antioxidant activities compared to the separated extracts/fractions of this resin. Thus, we suggest that the specific strong biological activities of the resin depend on the synergistic effect of all resin components when it is used completely as methanolic solution. These findings confirm the traditional preparation mode that uses whole crude pure resin as powder and confirm the traditional use of the resin for treatment of skin diseases due to infection with dermatophytes in the tropical island Soqotra.

Ethnobotanical Survey
The interview was conducted during February-March 1990 in ten different localities in Soqotra Island: Hadibu, Momi, Qalansieh, Haggeher, Noged, Mori, Qubah, Qadheb, Dihamidh and Serhin. 100 informants were interviewed (male 90; female 10). Most informants (80%) were within the age group of 35-60 years old who worked in the village. 20% of the informants were students in primary schools between 12-16 years. All informants were indigenous people; they spoke the local Soqotri language and Arabic. Informants had to identify the plant in the nature or the specimen carried during the survey.

Plant Material
Dracaena cinnabari plant parts were collected in their natural habitat. The plant was identified at Pharmacognosy Department, Aden University, Yemen. A voucher specimen (No. MF-SOC 001) has been deposited at the Herbarium of the Pharmacognosy Department, Pharmacy Faculty, Aden University, and at the personal Herbarium of the first author.

Data Analysis
Data generated from the field survey on the use of D. cinnabari was subjected to descriptive statistics using percentages and frequencies. To quantify the data generated from this survey, the following indices were used: use value index and fidelity level values.

Use Value (UV)
The use value [28,29] is a quantitative method that provides the relative importance of D. cinnabari by the local people in Soqotra Island. It was calculated according to the following formula: where UV refers to the use value of a species; U to the number of citations per species (D. cinnabari) given by informants; and N to the total number of informants.

Fidelity Level (FL)
Fidelity Level (FL) was calculated to determine the percentage of informants that reported the uses of D. cinnabari as a remedy for the same major ailment using the formula [17]: where Np is the number of informants who independently indicated the use of a species for the same major ailment and n the total number of informants who mentioned the plant species for any major ailment [18].

Samples of Standard Rresin and Adulteration Types of Resin
Two types of resins were collected from the endemic wild plant Dracaena cinnabari on slopes of Haggeher at Soqotra Island, Yemen, in February 1990 ( Figure 1). The superior fresh resin was collected from the young stems of the female tree, and stored as standard authentic resin. The alternative and inferior resin was found on the market in Soqotra Island, it was collected from old stems and mixed with small bark pieces.
The reference sample of pure Soqotri resin "Emzoloh" of wild D. cinnabari was collected from Soqotra Island. The market samples under the name "Dam Alakhwin" = Dragon's blood were purchased at local markets and suppliers in Soqotra Island, Aden city and Sana'a city. Resin samples were collected from markets in Soqotra Island, Aden and Sana'a. All market samples that were sold under the name of "Dam Alakhwin" were compared with authentic standard resin from Soqotra. The taxonomical and organoleptic methods [20] were used for identification of the pure resin or the adulterants.

Extraction of Resin for Biological Tests
The dried pure resin was pulverized in a grinder. 1 g pulverized resin was dissolved completely in 100 mL methanol. This methanol solution (MS) was kept as raw resin solution.
30 g pulverized resin was extracted with a mixture of 10% H 2 O/methanol. This mixture was partitioned against n-hexane (H), dichloromethane (D) and ethyl acetate (E), respectively. Extracts were filtrated and concentrated under reduced pressure at 40 • C. The resin residue remained after extraction was evaporated and dried. The residue was dissolved in methanol (MF) to use for biological evaluation.
Solution and extracts were stored in an exsiccator until use for antifungal, antioxidant and cytotoxicity tests.

Solubility Values
The solubility of the resin in different polar and non-polar solvents was determined according to the solubility test in the pharmacopeia [19].

Extractive Values
n-Hexane, dichloromethane, ethyl acetate, methanol and water-soluble extractive values were determined to find out the amount of polar and non-polar soluble compounds, using two types of the resin that were collected [20].

Antifungal Test (Agar Diffusion Test)
Antifungal activities of the extracts (2 mg dried extract per disc) were determined applying the agar diffusion assay [30]. Nystatin was used as positive control. Inhibition zone diameters include diameter of the disc (6 mm). The test organisms: Candida krusei (ATCC 90878), Absidia corymbifera

Determination of Antioxidant Activity
Qualitative and quantitative estimation of radical scavenging effect were carried out by 1,1-diphenyl-2-picryl-hydrazyl (DPPH) assay. Quantitative estimation was carried out according to the method of Brand et al. [16,31]. Ascorbic acid was used as positive control.
The reaction mixture contained 500 µL of test extract and 125 µL of DPPH in ethanol. Different concentrations of test samples (10, 50, 100, 500 and 1000 g/mL) were prepared while the concentration of DPPH was 1 mM in the reaction mixture. These reaction mixtures were taken in Eppendorf tubes and incubated at 37 • C for 30 min, the absorbance was measured at 517 nm. Percent radical scavenging activity by sample treatment was determined by comparison with ethanol treated control group. Ascorbic acid was used as positive control. The DPPH radical concentration was calculated using the following equation: Scavenging activity (%) = [(Absorbance control − Absorbance sample)/Absorbance control] × 100

Determination of Cytotoxicity Activity
The cytotoxicity was measured by the neutral red uptake assay [16,32], using FL-cells, a human amniotic epithel cell line. Only living cells are able to manage the active uptake of neutral red. FL-cells were cultivated in a 96-well microtiter plate (105 cell/mL EAGLEMEM, Sifin, Berlin, D, 150 L/well) at 37 • C in a humidified 5% carbon dioxide atmosphere. The EAGLE-MEM was completed by L-glutamin (0.10 g/L), Hepes (2.38 g/L), penicillin G (105 IE/L), streptomycin sulphate (0.10 g/L) and FCS (Gibco, 80.0 mL/L). After 24 h 50 L of the extract solution (extract dissolved in ethanol under stirring in ultrasonic bath for 5 min and then diluted with MEM: test mixture) or medium with equal amounts of ethanol (control) were added. After a further incubation for 72 h cells were washed three times with phosphate buffered saline solution. One hundred microliters of neutral red solution (SERVA, 0.3% in EGLE-MEM) were added per well. The cells were then incubated for 3 h at 37 • C, followed by another three times washing with PBS. One hundred microliters of a solution of acetic acid (1%, v/v) and ethanol (50%, v/v) in distilled water were added. After shaking for 15 min the optical density was measured at 540 nm with an ELISA-Reader HT II (Anthos Labtec Instruments, Salzburg, Austria). The mean of four measurements for each concentration was determine (n = 3).
Author Contributions: M.A. designed the overall project, performed experiments, data analysis, and wrote the manuscript.
Funding: This research received no external funding.