Gall Nuts Cynips quercusfolii (Linnaeus) and Andricus infectorius (Hartig) as Tannin Raw Materials

Abstract

The study included gall nuts caused by Cynips quercusfolii collected from Quercus robur and Andricus infectorius, harvested from Quercus infectoria. The evaluation of the percentage content of tannins expressed as a pyrogallol was performed using the analytical methodology recommended by the current “European Pharmacopoeia 10”. In order to compare the chemical composition of the tested samples, chromatographic profiles and ion mass distribution spectra were made using high-performance liquid chromatography techniques coupled with two types of detection: spectrophotometry and mass spectrometry. The average content of tannins expressed as pyrogallol for C. quercusfolii ranged from 13.36% to 14.74%, while for A. infectorius it was from 34.77% to 39.95%. The comparison of both the mass spectra and the chromatographic profiles shows a high similarity of all samples of C. quercusfolii and large differences in the chemical composition compared to A. infectorius. The tested C. quercusfolii should be considered a much poorer tannin source than A. infectorius. It cannot be simultaneously treated as its substitute raw material due to the lower tannins percentage content and different chemical composition. However, compared to other Central European herbal raw materials, C. quercusfolii gall nuts are an alternative tannin source with potential use in herbal medicine and the pharmaceutical industry.

1. Introduction

Quercus infectoria G. Olivier is a relatively small tree that forms groves under natural conditions of Greece and Asia Minor. It is there that Andricus infectorius (Hartig) lay their eggs on oak shoots. The larvae of these insects lead to excessive growth of the meristematic tissue of the buds, which results in the formation of very characteristic gall nuts. Gall nuts, as forms of extending the plant phenotype, are an example of manipulation and reprogramming of plant development by an alien (parasitic) organism, as a result of which spectacular morphological and physiological changes occur in plant tissues infested with insects [1,2]. Biochemical processes taking place in the attacked plants lead to the occurrence and accumulation of specific chemical compounds in gall nuts, such as phenolic acids, anthocyanins, flavonoids, tannins, steroids, triterpenes, alkaloids and saponins [3,4]. Therefore, gall nuts have been an area of focus for centuries. This interest has been growing recently, which is reflected by works of both cognitive and implementation character [5,6].

Strong healing properties of gall nuts collected from Q. infectoria have been known since antiquity. They were used in folk medicine to treat a wide range of diseases, among others, edema, inflammation, mouth infections, acute diarrhea and bleeding; they were also used as analgesics [7,8,9,10]. The results of modern research are in line with these centuries of experience. Currently, Q. infectoria gall nuts are believed to have antiviral, antibacterial, antifungal and antiparasitic properties [11]. They can be used in the treatment of nosocomial infections, periodontitis and tooth decay [3]. They also demonstrate a very high antioxidant activity [10]. The aforementioned directions of action result from the fact that gall nuts from Q. infectoria are a raw material particularly rich in tannins [10].

Gall nuts also occur on oaks in Central European forests, and both historical postulates [12,13,14], as well as the results of modern pilot studies [15,16], carried out on raw material from mid-field trees, show that they can be used in herbal medicine and the pharmaceutical industry. Although oaks (Quercus sp.) are very susceptible to parasitization by gall wasps [3], and C. quercusfolii and A. infectorius may occur on oaks in Central Europe [17], a certain problem for large scale collecting of Central European oak gall nuts is the lack of information in the available literature on their frequency of occurrence. It may stem from the fact that the appearance of these pathological outgrowths does not affect the condition of the trees. Gall wasps are, therefore, not of economic importance as pests in forestry. They are only one of the most fascinating and puzzling examples of parasitic symbiosis found in nature [1,18].

The suitable pharmacopoeial raw material [19] is the so-called Turkish galls, caused by A. infectorius and collected from the oak of Q. infectoria, harvested in the fall before complete insect development. This raw material was already described in renaissance medical sources, which recommended it, i.e., for sprinkling on wounds and inflammatory lesions in mucous membranes, relieving toothache and treating diarrhea [20,21]. A. infectorius gall nuts (Galla) also became part of official medicine in the 20th century, appearing in “Polish Pharmacopeia II” [22], “Polish Pharmacopeia III” [23], “Polish Pharmacopeia IV” [24], and after more than half a century a break-in “Polish Pharmacopeia XI” [25] and “Polish Pharmacopeia XII” [20], according to which A. infectorius gall nuts are the raw material for the production of gall nuts tincture (Gallae tinctura). In Poland, the most important species that are a potential source of gall nuts are the pedunculate oak (Quercus robur L.) and the less numerous sessile oak (Quercus petraea (Matt.) Liebl.), nowadays often treated as a subspecies of Q. robur [26]. Fresh broadleaved forest, moist broadleaved forest and moist mixed broadleaved forest are particularly suitable types of forest habitats for pedunculate oak, while fresh mixed broadleaved forest and moist mixed broadleaved forest for sessile oak [27]. A. infectorius [17] is theoretically possible in all these habitats. Unfortunately, the presence of A. infectorius in Poland is uncertain [28], but the formation on oak leaves by Cynips quercusfolii Linnaeus is very common.

Taking into account the above conditions, as well as the constantly growing share of oak stands in Polish forests, the potential raw material base of gall nuts is noteworthy and fits in with the concept of non-wood forest product use [15,29,30,31].

The aim of the study was to determine the content of tannins in Cynips quercusfolii gall nuts depending on the forest habitat type and in Andricus infectorius gall nuts depending on the date of their harvest. At the same time, an attempt was made to evaluate gall nuts of both species in terms of their suitability as a tannin raw material.

2. Materials and Methods

2.1. Raw Material Collection

In order to obtain gall nuts, the oaks growing in Głogów Małopolski Forest District (S-E Poland) were inspected. C. quercusfolii gall nuts were harvested from leaves of the pedunculate oak Q. robur. Three parts of material were collected for the study in three forest habitat types: moist mixed broadleaved forest—compartments 186b, 187d, 194d, fresh broadleaved forest—compartments 153a, 164a, 224a, moist broadleaved forest—compartments 140a, 169i, 170i. Depending on the place of collection, the number of collected gall nuts ranged from 31 to 140 pieces, with an average value of 76 pieces. The collected gall nuts were then dried at 40 °C in a PC DR 1116 forced air herb dryer (ProfiCook). The weight of the batch, depending on the place of harvest, after drying was from 6.8 to 52.8 g, with the average value for one batch being 24.7 g. Then, all the gall nuts were weighed, and the average mass of one gall was determined to be 0.326 g, gall nuts from each batch with a mean weight of ± 20%, i.e., in the range from 0.261 to 0.391 g were taken for grinding. The selected gall nuts were ground in an analytical mill (Carlstein) and sieved through a sieve with a mesh diameter of 0.18 mm (Multiserw), obtaining nine batches of the right raw material for testing.

For comparative purposes, tests were carried out on the pharmacopoeial herbal material of Q. infectoria oak shoots infected with A. infectorius, supplied from Turkey. These gall nuts were harvested according to the traditional procedure in the Southeastern Anatolia Region (historically known as a Mesopotamia, Kurdish populated area mostly) in two stages of development: in August, while the insects are still in the gall nuts and the gall nuts are green, and in September, after the insects had left the galls when the gall nuts became white-brown. The gall nuts from the first harvest date (green) were dried to a constant weight in the shade and in the air, while those from the second harvest date (white and brown) did not require drying. Then, the gall nuts were ground in an industrial mill EM1338 (Lavion, Turkey) and sieved through a sieve with a mesh diameter of 0.18 mm (Multiserw, Brzeźnica, Poland), thanks to which two consecutive batches of raw material were obtained for testing.

2.2. Determination of Tannin Content

In order to assess the tannin content of Q. robur oak and Q. infectoria oak, an analytical methodology with hide powder was used, reflecting the pharmacological properties of the raw material, i.e., binding to and precipitating protein. This protein is hide powder, specifically, ground pure collagen. The research methodology consists of determining the percentage content of tannins by calculating the difference between Total polyphenols and Polyphenols not adsorbed by the hide powder while referring to the standard, i.e., pyrogallol solution. This methodology uses the reaction of polyphenols with phosphomolybdotungstic reagent in which, in the sodium carbonate environment, after 30 min of incubation, blue reaction products are formed. The intensity of the blue color of the solution is proportional to the polyphenol content and is tested with a spectrophotometer by measuring the absorbance at 760 nm, using water as the compensation liquid. Due to the instability of tannins in aqueous solutions, all the extraction and dilution operations must be protected from light. This methodology is recommended by the latest “European Pharmacopoeia 10” [32] and was previously used in our earlier works [15,16]. The research used a 356P water bath (Unipan, Warsaw, Poland), MST magnetic stirrer (Velp Scientifica, Usmate Velate, Italy), analytical and semi-analytical balances (AS110.R2 Radwag, Radom, Poland, Vibra DJ-150S Shinko Denshi, Vibra SJP Shinko Denshi, Tokyo, Japan) and a Vis 5100 spectrophotometer (Metash, Shanghai, China). The following reagents were used: pyrogallol (Sigma, Aldrich, St. Louis, MO, USA), hide powder (EDQM, Strasbourg, France), sodium carbonate (Stanlab, Lublin, Poland), 0.45 µm syringe filters (Whatman and Merck, Darmstadt, Germany) and purified water with a conductivity below 0.05 µS/cm (Basic 5, Hydrolab, Straszyn, Poland). The obtained results were expressed in accordance with the pharmacopoeial recommendation as a pyrogallol.

Both the chromatographic profiles and the ion mass distributions of the A. infectorius and C. quercusfolii gall nuts were obtained by using the high-performance liquid chromatography (HPLC) technique coupled with two types of detection: spectrophotometry using a photodiode array detector (DAD) (Agilent Technologies 1200 Series, Waldbronn, Germany) and mass spectrometry (triple quadrupole MS, 6470 Agilent Technologies Waldbronn, Germany). The obtained chromatograms were recorded at a wavelength of 276 nm. Ion mass distribution spectra were obtained in the SCAN mode of MS operation. Detailed operating parameters of the chromatographic system are presented in

Table 1. Characteristics of parameters for the determination of tannin compounds by means of the HPLC-DAD-MS technique.

Parameter	Characteristics
Stationary Phase
Type, manufacturer	Poroshell 120 EC-C18 (Agilent Technologies, Santa Clara, CA, USA), 4.6 × 150 mm, 2.7 µm
Column temperature	T = 25 °C
Mobile Phase
Phase composition	A: water, 5 mM ammonium formate in water (99.9%), 0.1% formic acid (0.1%) B: methanol, 5 mM ammonium formate in methanol (99.9%), formic acid (0.1%)
Flow rate	Vflow = 0.5 mL/min
Gradient Elution Profile	0 min–80% A, 20% B 0.1–10 min–50% A, 50% B 10.1–14 min–10% A, 90% B 14.1–20 min–80% A, 20% B
Detector with a photodiode array (DAD) 276; 8 nm, Ref. 360, 100 nm
Mass spectrometer detector (MS): electrospray (ESI), ionization mode: [M-H]−, fragmentor 135, carrier gas flow rate 5 L/min, carrier gas flow temperature 350 °C, voltage on the capillary 3500 V, mass range 50–2000 (m/z)
Analysis time	20 min
Sample injection volume	5 µL

.

The study was preceded by the preparation of standard solutions of gallic acid and tannic acid—tannins (Sigma-Aldrich, St. Louis, MO, USA), consisting of dissolving certified standards of both substances in water and obtaining solutions with a concentration of 2 and 9 mg/mL, respectively. Real samples were prepared by suspending about 20–30 mg of powdered real samples in 50 mL of deionized water, followed by a half-an-hour extraction in a boiling water bath. The obtained extracts were analyzed with HPLC-DAD-MS.

2.3. Statistical Analysis

With regard to the study on the impact of the forest habitat type on the tannin content in C. quercusfolii gall nuts and the effect of the harvest date on the tannin content in A. infectorius gall nuts, a one-way analysis of variance (one-way ANOVA) was performed using R software. To find means that are significantly different from each other, a post-hoc analysis with Tukey’s Honest Significant Differences test was used. The differences were deemed statistically significant with a cut-off level alpha = 0.05.

3. Results

The average content of tannins expressed as pyrogallol in Central European raw material (C. quercusfolii), depending on the habitat type of forest, ranged from 11.86% to 14.50% for the moist mixed broadleaved forest, from 12.95% to 15.11% for fresh broadleaved forest and from 14.01% to 15.95% for a moist broadleaved forest (

Table 2. Content of tannins in Cynips quercusfolii gall nuts.

Forest Habitat Type	Forest Compartment	Tannins Content [%]									Tannins Content-Average per Forest Site [%]
Moist mixed broadleaved forest	194d	12.16	12.09	12.12	11.86	11.91	11.92	12.27	12.31	12.24	13.36
	187d	13.77	13.58	13.52	13.85	13.98	14.02	13.54	13.44	13.24
	186b	14.42	14.50	14.38	14.34	14.29	14.22	14.25	14.16	14.31
Fresh broadleaved forest	224a	14.75	14.76	14.55	15.05	15.11	14.95	14.83	14.79	14.90	14.06
	164a	13.22	13.10	13.15	13.08	13.09	12.95	13.23	13.40	13.34
	153a	14.24	14.32	14.26	14.19	13.99	14.10	14.11	13.97	14.30
Moist broadleaved forest	170i	15.56	15.61	15.42	15.95	15.80	15.92	15.79	15.84	15.89	14.74
	169i	14.18	14.25	14.21	14.27	14.29	14.11	14.36	14.40	14.39
	140a	14.12	14.10	14.01	14.11	14.28	14.19	14.29	14.19	14.42

). Statistically significant differences between the mean values of tannin content between all the different habitat types of the forest were shown (Figure 1A). In turn, for A. infectorius gall nuts, the tannin content ranged from 34.39% to 35.21% for the first harvest and from 38.70% to 41.09% for the second harvest (

Table 3. Content of tannins in Andricus infectorius gall nuts.

Harvest Date	Tannins Content [%]	Tannins Content- Average per Term of Collection [%]
I (August)	34.59	34.77
	34.39
	34.60
	34.74
	34.62
	34.70
	34.95
	35.11
	35.21
II (September)	40.47	39.95
	41.09
	40.85
	39.92
	39.87
	39.90
	38.70
	38.91
	39.85

). In this case, the analysis also confirmed statistically significant differences (Figure 1B).

Tannins are natural plant substances with a high molecular weight, from 300–5000 Da. Profiling of the content of tannic acid, considered to be the most important active ingredient of oak galls, is determined by examining the content and proportion/ratio of gallotannins [32,33,34,35]. Initially, a direct injection of aqueous extracts of gall nuts and gallic and tannic acid standards (GA and TA) was made to the mass spectrometer in order to check the ion mass distribution in the tested samples. Figure 2 shows the result of this analysis.

The distribution and quality profile of the masses in A. infectorius gall nuts largely coincides with the masses characterizing the mixture of standards (gallic and tannic acid), the same values (m/z) of ions obtained in the SCAN mode [M-H]⁻ appear, e.g., 169, 247, 321, 939, 1091, 1396, which proves that these gall nuts contain compounds in the same ionic form as in the gallic and tannic acid standards (Figure 2A).

A. infectorius gall nuts are rich in galloyl glucoses (from mono to octa-galloylglucose), compounds characteristic of the MS spectrum of tannic acid (Figure 2C). This is evidenced by the values of (m/z) [M-H]⁻ ions visible in MS spectra (169 gallic acid, 321 digallic acid, 33 galloylglucose, 483 digaloyl glucose, 635 trigaloyl glucose, 787 tetragaloyl glucose, 939 pentagaloyl glucose, 1091 hexagaloyl glucose, 1243 heptagaloyl glucose, 1396 octagaloyl glucose). In turn, C. quercusfolii gall nuts contain quinic acid (in the [M-H]⁻ MS spectrum); this is the parent ion with 191 mass (m/z) (Figure 2B). These gall nuts contain parent ions with masses indicating the presence of ellagic acid 301 (m/z) and gallic acid 169 (m/z). These gall nuts are relatively poor in tannin compounds in the form of galloyl glucoses.

The chromatographic profiles (DAD, MS) are a kind of “tannin fingerprint”, clearly showing the profile of the tannin compounds in individual samples. The chromatograms (HPLC-MS-SCAN) (Figure 3) show the distribution of tannic compounds, galloyl glucoses in an aqueous solution of tannic acid, and in samples of Polish and Turkish gall nuts, they elute from 5 to 15 min, immediately after gallic acid (tr = 4 min). As the retention time increases, the mass of detected parent ions increases (from 469 to 1396 m/z).

The above is confirmed in more detail by the chromatograms of the gall nut samples obtained during the HPLC-DAD analysis.

By analyzing the chromatograms presented in Figure 4, it can be concluded that A. infectorius (A, B, C) and C. quercusfolii (C, D) gall nuts differ significantly in their profile, i.e., composition and amount of tannic compounds: gallic acid content (tr = 4 min) and gallotannins (tr = 5–15 min), while within one forest habitat type and also between the three studied habitat types of forests, the differences are small. Additionally, taking into account the sum of the areas of all peaks in the chromatograms for individual samples of C. quercusfolii gall nuts from a given forest habitat type, it can be seen that the average content of tannins in Polish gall nuts from a moist broadleaved forest is the highest. On the other hand, within A. infectorius gall nuts, a greater total tannin content was observed in the sample from the second harvest, which confirms the results obtained with the hide powder method.

On the basis of the available values of the areas on the chromatograms for individual peaks (compounds), the percentage share of gallic acid in the water extracts of gall nuts was also calculated in relation to the total amount of determined compounds (total area of all peaks) (HPLC-DAD). The average content of gallic acid in Polish gall nuts is 3.30% (ranges from 2.85% to 3.97%), while Turkish gall nuts contain 5.04% and 3.85% for the first and second harvest, respectively.

Interestingly, HPLC-DAD chromatograms (Figure 3 and Figure 4A–C) and the mass spectra (Figure 2B) demonstrate that Polish C. quercusfolii gall nuts are rich in quinic acid (QA, quinic acid, rt approx. 3 min, 191 m/z), the average content of which is 9% of all compounds present in the chromatogram. Turkish gall nuts contain only about 1% of this acid (Figure 2C, Figure 3 and Figure 4D,E).

4. Discussion

According to the pharmacopoeial definition, the so-called Turkish galls, caused by A. infectorius, are greenish-brown to yellow-brown in color. Thus, both gall nuts with a hollow indicating that A. infectorius larvae have left the gall nuts and also dried gall nuts with the remains of dead insects are classified as pharmacopoeial raw materials. For this reason, this study includes both gall nuts from the first harvest date, i.e., green without a hollow, and yellowish ones with a hollow. The minimum content of tannins expressed as pyrogallol, standardized for this raw material, is at least 20.0% [24], and it must be analyzed with the so-called hide powder method, using the spectrophotometric technique, for which the analytical procedure has been described in detail by the current “European Pharmacopoeia” and also the “Polish Pharmacopoeia” [19,25]. Such a technique was used in this study. Meanwhile, most of the studies published so far, devoted to both A. infectorius gall nuts and C. quercusfolii galls, present the results obtained using analytical methods other than those recommended by the current pharmacopoeia [19,25]. In Paaver et al.’s work [5], in which Price and Butler’s method was used, an average tannin content was found at the level of 81.4% for Turkish galls, and the results were also given for other herbal tannin raw materials, such as oak bark (Quercus cortex) and torrmentil (Tormentillae rhizoma), making it visible that the applied methodology overestimates the content of tannins by 2–3 times in relation to the results obtained for the hide powder method [36,37]. In our study, the average tannin content expressed as pyrogallol was 34.77% for the first harvest date and 39.95% for the second harvest date. The minimum pharmacopoeial standard (20.0%) is, therefore, significantly exceeded for the tested raw material, which suggests the need to conduct more extensive studies on Turkish gall nuts and a possible revision of the pharmacopoeial monograph [25].

In this study, the composition of A. infectorius gall nuts from Turkey using chromatographic and mass spectrometric techniques was compared with the composition of C. quercusfolii gall nuts obtained from the Polish forest environment. Contrary to A. infectorius gall nuts, the gall nuts of Central European oaks, formed as a result of leaf puncture by C. quercusfolii, have never been a pharmacopoeial material but have been widely used in folk medicine under the names of oak-galls, galls or oak apples. The indications for their therapeutic use were very broad and similar to those attributed to A. infectorius galls, covering numerous diseases for which, in the light of modern medicine, tannins can be effective. These were skin diseases (in the form of squeezed juice) [38], diarrhea (in the form of a tincture) [39], wounds [40] and toothache [41]. The high efficiency of C. quercusfolii gall nuts in folk medicine has attracted the attention of scientists for a long time. Already at the beginning of the 19th century, the possibility of replacing Turkish gall nuts with native ones was suggested, noting its less effectiveness as a raw material but its common incidence and, therefore, easier and less expensive collecting practice [12]. However, only a few scientific papers were published devoted to C. quercusfolii gall nuts as a possible alternative to A. infectorius galls. Both Sokołowska [13,14] and Paaver et al. [5] indicate their high potential usefulness in the pharmaceutical industry. On the other hand, Bilek et al. [15,16] studied C. quercusfolii gall nuts collected at various dates and of various sizes, with the raw material coming from mid-field trees. At the same time, a differentiated content of tannins, ranging from 10.0% to 15.8% expressed as pyrogallol, was demonstrated, and it was emphasized that C. quercusfolii gall nuts are the richest Central European tannin material [24].

In this study, it was found that the average content of tannins expressed as pyrogallol, depending on the three habitat types of forest, differs significantly and ranges from 13.36% for the moist mixed broadleaved forest to 14.74% for the moist broadleaved forest. However, the values for all three forest sites confirm the previous study results, which place C. quercusfolii gall nuts as the richest tannin raw material that could be obtained in Central Europe [15,24]. However, a comparison of their qualitative composition, determined on the basis of HPLC-DAD-MS (“fingerprint”) chromatographic profiles, shows significant differences with A. infectorius gall nuts (Figure 3 and Figure 5). Further, the total tannin content in A. infectorius gall nuts is two to three times higher, which is indicated, in turn, by a study performed with the hide powder method using spectrophotometric techniques.

Similar to the results obtained with the hide powder method, also comparing the total ion mass distribution profiles (direct injection of the sample to the MS detector) of C. quercusfolii gall nuts (Polish galls) with A. infectorius gall nuts (Turkish galls), it can be concluded that Turkish gall nuts are a valuable source of tannins (mainly galloyl glucoses), while Polish gall nuts contain much less of them, including gallic acid (Figure 2 and Figure 3).

The most important thing is, however, that the obtained results (hide powder method, HPLC-DAD-MS SCAN) clearly show that Polish gall nuts contain much fewer tannin compounds compared to the composition of Turkish galls. Thus, both the composition and the total tannin content indicate that C. quercusfolii gall nuts cannot be a substitute for A. infectorius galls but still remain a very valuable and the richest tannin raw material in this part of Europe.

For this reason, the potential use of gall nuts may be analogous to that of other tanning-rich raw materials, e.g., tormentil, oak bark or raspberry leaf. The European Medicines Agency has approved the traditional use of herbal medicinal products from these raw materials, including in the form of comminuted/powdered herbal substances or tinctures. Regardless of the differences in the total tannin content and in their qualitative composition, they have analogous therapeutic indications, i.e., “symptomatic treatment of minor inflammations of the oral mucosa”; such pharmaceutical forms and therapeutic indications could also refer to the galls of C. quercusfolii. The registration of such herbal medicinal products would be easier if quality criteria were introduced in the current “European Pharmacopoeia” in the form of a pharmacopoeial monograph. The standards for the content of tannins in the gall nuts of C. quercusfolii, as well as the identification by mass spectrometry technique presented in this paper, could form the basis for its development.

5. Conclusions

• C. quercusfolii gall nuts are commonly occurring on Q. robur oaks. They can be harvested and used as a very rich tannin raw material.
• The tannin content of C. quercusfolii gall nuts depends on the forest habitat type; the highest concentrations were found for moist broadleaved forest.
• C. quercusfolii gall nuts should be considered a tannin material much poorer than A. infectorius gall nuts included in the current pharmacopoeia.
• Due to the lower tannin percentage content and the different chemical composition, C. quercusfolii gall nuts cannot be treated as a substitute material for A. infectorius galls.