Development of a Technology for Pharmaceutical Preparations from Nigella sativa Seeds and Their Quality Standardization

The study is devoted to developing a medicinal product based on Nigella sativa seeds that combines hydrophobic and polar bioactive substances, including essential fatty acids, thymoquinone, amino acids, phenolic and organic compounds, and mineral constituents. Particular attention is given to the phytochemical characterization of domestic raw material, which is comparable in its valuable bioactive substance content to imported material, and to the development of an accurate quantitative determination of thymoquinone.

The aim was to obtain a pharmaceutical substance by supercritical carbon dioxide extraction, create its soft gelatin encapsulated dosage form, and standardize its quality. The work covers the selection of extraction parameters, investigation of extract composition and reducing capacity, establishment of numerical quality indicators, microscopic analysis, and determination of storage conditions and shelf life.

INTRODUCTION

Relevance of the research topic. Seeds of Nigella sativa (NSS) are well known in folk medicine and are used in the treatment of a wide variety of diseases. The broad spectrum of Nigella use is attributable to its distinctive composition of bioactive substances (BAS), the most significant of which are essential fatty acids and thymoquinone. Experimental studies have demonstrated pronounced antioxidant and hepatoprotective effects of fatty acids and thymoquinone. The seeds' polar BAS—including amino acids, phenolic compounds, and organic acids, which predominantly have hepatoprotective and choleretic properties—also give them substantial value. Multigenerational experience with NSS in folk medicine, its rich chemical composition, and available information on its pharmacological properties place this plant raw material among the most important sources for obtaining pharmaceutical preparations for the treatment of hepatobiliary system disorders.

The only product of NSS processing is fixed oil (Black cumin oil, Nigella oil), which is used as a food dietary supplement (DS). The cold-pressing technology used to produce it enables only hydrophobic BAS groups, in particular fatty acids, to be concentrated in the finished product. The polar BAS remain in the cake and are not utilized. Moreover, one of Nigella's principal active components, thymoquinone, is present in the oil only in minor amounts. For these reasons, fixed oil from NSS cannot be considered a potential medicinal product derived from this plant raw material.

Given the value of both nonpolar and polar NSS constituents, the production of a pharmaceutical substance from them that combines the full range of necessary BAS is justified. It is also important to provide such a substance, taking account of its chemical composition and possible medical use, with an appropriate dosage form.

Thus, the development of a technology for obtaining pharmaceutical preparations—a pharmaceutical substance from NSS containing BAS of differing polarity and its dosage form—represents a relevant research topic in pharmacy.

Degree of development. At present, studies have produced results, and information on the pharmacological properties and phytochemical composition of Nigella sativa is presented in sufficient detail. However, these studies were not originally aimed at developing pharmaceutical preparations from this plant raw material. The difficulty of combining the necessary but polar-diverse BAS in a single pharmaceutical product also contributed to the absence of a medicinal product from NSS. Achieving such a combination with conventional technologies proved highly difficult. Not surprisingly, fixed oil from NSS, whose chemical composition is limited to hydrophobic BAS, does not reveal the full pharmacotherapeutic potential of this plant raw material and is used only as a food dietary supplement.

Goal and objectives. The aim of the present study was to develop a technology for obtaining a pharmaceutical substance from Nigella sativa seeds, its dosage form, and to standardize their quality.

To achieve this goal, the following tasks had to be addressed:

- to study the main groups of BAS and standardize the quality of Nigella sativa seeds, using the results obtained to develop pharmaceutical preparations from this plant raw material;

- to justify the use of supercritical carbon dioxide extraction for obtaining a pharmaceutical substance from Nigella sativa seeds;

- to develop a technology for obtaining a supercritical carbon dioxide extract of Nigella sativa oriented toward the leading BAS;

- to propose quality standards for the developed supercritical carbon dioxide extract of Nigella sativa and to study its storage conditions and shelf life;

- to justify the type, develop the composition and technology for obtaining a dosage form of the supercritical carbon dioxide extract of Nigella sativa, standardize its quality, and study its storage conditions and shelf life.

Novelty. The expediency of obtaining pharmaceutical preparations containing polar-diverse BAS from NSS for the treatment of hepatobiliary system disorders has been demonstrated. It has been established that the optimal pharmaceutical substance from NSS is a supercritical carbon dioxide extract, which differs favorably from the fixed oil of this plant raw material by containing not only hydrophobic BAS but also the necessary polar substances and, most importantly, by having an approximately tenfold higher content of the principal active substance—thymoquinone.

Rational technological regimes for supercritical carbon dioxide extraction have been determined for extracting active and other leading BAS from Nigella sativa seeds and ensuring their optimal quantitative content in the pharmaceutical substance. The ability to control the chemical composition of the pharmaceutical substance obtained from NSS, and accordingly to predict its pharmacotherapeutic properties, by varying the technological regimes of supercritical carbon dioxide extraction has been demonstrated. Quality indicators for the obtained supercritical carbon dioxide extract from NSS were proposed and standardized, and its storage conditions and shelf life were determined.

Taking account of the pharmacological and physicochemical properties of the constituent BAS and the technological features of the pharmaceutical substance from NSS, the composition and method for obtaining soft capsules of the supercritical carbon dioxide extract were developed. The quality of the developed dosage form was standardized, and its storage conditions and shelf life were established.

Capillary electrophoresis was used for the first time for the quantitative determination of thymoquinone. Its main advantage over the HPLC method used is rapidity and, in practice, the absence of a need to use expensive and highly toxic solvents. An electrophoretic method for the quantitative determination of thymoquinone in raw material and in medicinal preparations from Nigella sativa was proposed and validated.

Theoretical and practical significance. Using the technology for a pharmaceutical substance from NSS as an example, the possibility of using supercritical carbon dioxide extraction to obtain phytopharmaceutical preparations that combine polar-diverse BAS has been demonstrated. Knowledge has been expanded in the field of the technology for obtaining fluid phytoextracts, the process, and the features of the diffusion of BAS of differing polarity during supercritical carbon dioxide extraction. The results of studies of supercritical carbon dioxide extraction of Nigella seeds contribute to the implementation of this method for producing plant-derived medicinal products at specialized pharmaceutical enterprises. The proposed list of quality indicators and standards for the supercritical carbon dioxide extract from Nigella sativa seeds may be taken into account by developers of analogous pharmaceutical products.

Comparative phytochemical studies of NSS raw material harvested in Southern Russia and imported raw material showed comparable contents of valuable BAS and the possibility of using domestic raw material to obtain medicinal products.

Three enterprise pharmacopoeial article projects (FSPs) were developed on the basis of the research: Seeds of Nigella sativa (approval act of LLC PKF Fitofarm dated 12.01.2015); Supercritical Carbon Dioxide Extract of Nigella sativa and Soft Capsules of Supercritical Carbon Dioxide Extract of Nigella sativa 250 mg (approval acts of LLC ER Research Center GORO dated 11.02.2015 and 26.02.2015). The methods presented in the FSP projects were approved at the State Budgetary Institution of the Krasnodar Territory Pharmaceutical Center (approval act dated 03.02.2015). The technology for obtaining a supercritical carbon dioxide extract of Nigella seeds was approved at the Research Center of Ecological Resources GORO (approval act dated 29.12.2014). The technology for obtaining and methods for assessing the quality of a soft encapsulated dosage form containing a supercritical carbon dioxide extract from Nigella sativa seeds were approved at LLC INTEHPROEKT (approval act dated 30.12.2014). The dissertation research materials were introduced into the teaching process of the pharmacy department of Kuban State Medical University (KubSMU), Ministry of Health of Russia (introduction act dated 11.05.15), and pharmaceutical technology at Siberian State Medical University (SibSMU), Ministry of Health of Russia (introduction act dated 19.05.15).

Methodology and research methods. The work is based on a methodology for developing original combined phytopharmaceutical preparations through identification of active substances and orientation of the production and quality-standardization technology to those substances.

Pharmaceutical and biopharmaceutical analysis methods, as well as technological and mathematical methods, were used in the dissertation study.

The study design reflects the structure and sequence of all stages of the dissertation work.

Connection of the research with the pharmaceutical sciences research program. The dissertation was carried out within the research theme Creation of original combined phytopharmaceutical preparations and modernization of those used in medicine on the basis of a methodology for identifying active substances (state registration No. 01201250744).

Propositions put forward for defense:

- data on the study of the phytochemical composition of the main groups of BAS and the standardization of the quality of Nigella sativa seeds;

- justification for using supercritical carbon dioxide extraction to obtain a pharmaceutical substance from Nigella sativa seeds;

- results of the study on developing a technology for obtaining a supercritical carbon dioxide extract of Nigella sativa and establishing its quality standards;

- results of the study on justifying the type, developing the composition and technology for obtaining a dosage form of the supercritical carbon dioxide extract of Nigella sativa.

Reliability and approval of the work. The reliability of the results obtained in the dissertation was confirmed by the use of modern physical, physicochemical, chemical, technological, biopharmaceutical, and mathematical methods. The results of the studies were subjected to statistical processing.

The dissertation materials were presented at the All-Russian Scientific and Practical Conference with International Participation dedicated to the 45th anniversary of the Pharmacy Faculty of Kursk State Medical University (Kursk, 2011); the 1st All-Russian Scientific and Practical Conference of Young Scientists Problems of Developing New Medicinal Products (Moscow, 2013); the II International Scientific and Practical Conference Actual Problems of Medicine in Russia and Abroad (Novosibirsk, 2015); and the X International Scientific and Practical Conference Theoretical and Practical Aspects of the Development of Scientific Thought: Medical Sciences, Pharmaceutical Sciences, Veterinary Sciences, Biological Sciences, Chemical Sciences (Moscow, 2015).

Twelve works were published on the topic of the dissertation, including six articles in journals recommended by the VAK.

Author's personal contribution. The research results presented in the dissertation were personally obtained by the author. With the author's direct participation, phytochemical studies were conducted, the technology for medicinal products from NSS—the pharmaceutical substance and its encapsulated dosage form—was developed, and their quality was standardized. The author is the principal developer of the normative documentation for Nigella seeds, the supercritical carbon dioxide extract, and soft capsules containing it, and is also the lead performer in the writing of scientific articles.

Volume and structure of the dissertation. The dissertation is presented in 170 pages of computer-set text and consists of a literature review, four chapters of original research, general conclusions, a conclusion, and a list of 201 sources, including 86 foreign-language sources; it contains 40 tables and 29 figures.

CHAPTER 1 CURRENT STATE OF RESEARCH ON THE CHEMISTRY, PHARMACOLOGY, AND APPLICATION OF NIGELLA SATIVA (LITERATURE REVIEW)

Nigella sativa L. (black cumin, cultivated cumin, kalinja, nigella, Roman coriander, cumin) is an annual herbaceous, light-green, slightly glaucous plant of the family Ranunculaceae, with an erect, branched stem 15–50 cm high. The seeds have a pepper taste and a musky aroma owing to the essential oils they contain [37, 163].

Nigella sativa is currently grown in southeastern Bulgaria and North America, and is cultivated in Central and Southern Europe, Eastern and Southern Asia, the western Mediterranean, the Middle East (Pakistan, Afghanistan, Saudi Arabia, and Iran), North Africa, primarily Tunisia, and India. In addition, Nigella sativa is widespread in Lithuania, southern and western Ukraine, Moldova, and Transcaucasia [37, 47, 85, 120, 135].

Nigella sativa is also widely distributed in the Russian Federation. In particular, it grows both wild and cultivated in Crimea, the Stavropol and Krasnodar Territories, Rostov Oblast, and the Republic of Adygea [7, 37, 51]. Introductory studies are currently being conducted to introduce Nigella sativa into cultivation under the conditions of mountainous Dagestan [109]. In addition, Nigella occurs in European Russia [37].

Thus, the reserves of this plant are concentrated primarily in Southern Russia. Consequently, the issue of the raw-material base and, accordingly, the replacement of imported plant raw material with domestic material—a source for obtaining potential medicinal products from Nigella sativa—can be resolved in the Russian Federation without particular difficulty.

1.1 Results of Studies of the Chemical Composition of Nigella sativa

For many decades, especially in recent years, growing interest has been noted among foreign and domestic scientists in studying the chemical composition of Nigella sativa and, above all, its seeds. The seeds are the richest source of fixed oil (black cumin oil, nigella oil) [50, 69, 112, 139]. The value of black cumin fixed oil is due to its distinctive composition of diverse groups of lipophilic BAS, among which fatty acids have particular significance (Table 1) [151, 164, 175, 192].

Table 1 — Fatty acid composition of black cumin oil

Trivial name | Systematic name | Gross formula

Saturated fatty acids

Myristic acid | Tetradecanoic acid | C13H27COOH

Palmitic acid | Hexadecanoic acid | C15H31COOH

Stearic acid | Octadecanoic acid | C17H35COOH

Arachidic acid | Eicosanoic acid | C19H39COOH

Behenic acid | Docosanoic acid | C21H43COOH

Lignoceric acid | Tetracosanoic acid | C23H47COOH

Margaric acid | Heptadecanoic acid | C16H33COOH

Pentadecanoic acid | Pentadecanoic acid | C14H29COOH

Monounsaturated fatty acids

Myristoleic acid | cis-9-tetradecenoic acid | C13H25COOH

Palmitoleic acid | cis-9-hexadecenoic acid | C15H29COOH

Oleic acid | cis-9-octadecenoic acid | C17H33COOH

Polyunsaturated fatty acids

Linoleic acid | cis,cis-9,12-octadecadienoic acid | C17H31COOH

Linolenic acid | cis,cis,cis-6,9,12-octadecatrienoic acid | C17H28COOH

Arachidonic acid | cis,cis,cis-5,8,11,14-eicosatetraenoic acid | C19H31COOH

The data on the chemical composition of black cumin oil presented in Table 1 show that it consists of saturated, monounsaturated, and polyunsaturated fatty acids. It has also been established that essential fatty acids constitute one of the leading groups of BAS in the hydrophobic fraction of Nigella sativa seeds, accounting for their use in folk medicine [70, 200].

According to [50, 140, 145, 172], linoleic acid (55–65%), oleic acid (15–18%), and palmitic acid (10–12%) occupy leading positions in the fatty acid composition of the oil. Because linoleic acid predominates in black cumin oil, the oil belongs to the group of semi-drying oils. Glycerol, selinene, benzoic acid, and phenylacetic acid have also been identified in black cumin oil. Several researchers [50] have determined the quantitative content of black cumin fixed oil, which varies from 28.0% to 36.44%. As for the content of essential fatty acids in Nigella sativa seeds, chromatography-mass spectrometry made it possible to determine this indicator at 32.53% [50].

The study of non-saponifiable BAS in black cumin fixed oil revealed the presence of dehydroretinol acetate, tocopherol acetate, and methylretinol acetate [50, 164]. As for the carotenoids and chlorophylls present in the fixed oil, the literature lacks exhaustive information on the quantitative composition of these groups of BAS, which make a partial contribution to the therapeutic activity of this plant raw material. The phospholipids of black cumin oil are represented by phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol [112].

An important group of bioactive substances present in black cumin fixed oil, in addition to the compounds considered above, is sterols, whose content ranges from 1993 mg/kg to 2887 mg/kg. The sterol composition of black cumin oil includes campesterol, sitosterol, stigmasterol, cholesterol, alpha-spinasterol, beta-sitosterol, 5-avenasterol, and 7-avenasterol [164, 183].

It should be noted that several authors using HPLC reported the presence of one of the principal pharmacologically active compounds of this plant—thymoquinone (Figure 1)—in black cumin fixed oil, but in a very minor amount (0.02–0.05%) [174, 179]. The minimal transfer of thymoquinone into black cumin fixed oil is attributable to its physicochemical properties, and the traditionally used cold-pressing and circulatory extraction methods currently in use do not facilitate it.

Structural formula (A): 2-isopropyl-5-methylbenzo-1,4-quinone.

Gross formula (B): C10H12O2.

Figure 1 — Structural (A) and gross (B) formulas of thymoquinone.

In addition to fixed oil, Nigella seeds contain essential oil, the quantitative content of which varies from 0.46% to 1.5% [140]. Because of the small amount of essential oil, Nigella seeds are not considered an industrial source for its production. At the same time, the chemical composition of Nigella essential oil is enriched with diverse BAS, among which the aforementioned thymoquinone is the principal component; its content reaches about 25%, and according to some data is higher (about 57%) [141, 175].

Gas-chromatographic methods have also identified p-cymene, ethyl linoleate, alpha-pinene, ethyl hexadecanoate, ethyl oleate, beta-pinene, limonene, carvacrol, camphene, sabinene, myrcene, alpha-terpinene, beta-phellandrene, gamma-terpinene, terpinolene, linalool, borneol, terpinen-4-ol, 1,8-cineole, camphor, carvone, bornyl acetate, longifoline, alpha-longipinene, beta-thujone, p-cymen-8-ol, trans-sabinene hydrate, cis-sabinene hydrate, (E)-anethole, artemisia ketone, 2-undecanone, ethyl hexanoate, ethyl heptanoate, ethyl octanoate, ethyl tetradecanoate, ethyl octadecanoate, methyl linoleate, 2-heptenal, ethyl nonanoate, thymohydroquinone, dipropyl disulfide, dibutyl disulfide, and butyl propyl disulfide in Nigella sativa seeds [141, 145, 168]. In addition, scientists have determined the presence of nigellone (nigellone) in the essential oil, which, according to some data, is a mixture of dithymoquinone and thymoquinone and, according to others, is dithymoquinone [120, 121, 124]. No quantitative characterization of nigellone in seeds is given. The most significant compounds of Nigella essential oil are considered to be thymoquinone, nigellone, and carvone, which determine the characteristic aromatic odor of its seeds [33, 188].

Analysis of data on the direct determination of thymoquinone content in Nigella seeds revealed limited information on the one hand and scattered information on the other. According to the studies, the thymoquinone content in Nigella sativa seeds varies from 0.06% to 0.5% and may reach 1.0% [179]. It should be noted that the qualitative and quantitative determination of thymoquinone was carried out by HPLC in all cases, a method with sufficient sensitivity and accuracy. The contradictory information on the quantitative content of thymoquinone in Nigella seeds is probably related, first, to geographical and climatic features of the raw-material growing sites and, second, to the use of various inorganic and organic solvents that differ in extraction ability toward the compound. This may also be related to data on its chemical properties—polarity—that have not yet been precisely established. The results reflected in [117] nevertheless indicate the ability of thymoquinone to dissolve in water (ratio: 0.87 mg per 1 mL at 25 °C). For example, [117] studied the solubility and stability of thymoquinone in aqueous solutions. The study showed that even in aqueous solutions, the extent of thymoquinone release (>500 µg/mL) is sufficient to achieve a therapeutic effect in vivo. However, aqueous solutions of thymoquinone were found to be unstable. These data correlate with tests confirming the unsuitability of thymoquinone as a pharmaceutical substance and in medicinal preparations in truly aqueous solutions. According to [179], however, thymoquinone demonstrates the greatest stability and extractability in aqueous-alcoholic solutions. The analysis presented in [116] shows results different from the foregoing and characterizes the hydrophobic properties of this substance to a greater extent.

Another possible reason for such a wide variation in the quantitative range of thymoquinone in Nigella seeds may be the ability of this substance to transform, through photodimerization, into thymohydroquinone under certain conditions [80, 145, 168]. This process is reversible. It has also been established that thymoquinone is a product of the oxidative degradation of thymol, which is formed through the intermediate thymohydroquinone. In addition, thymoquinone undergoes photodimerization to dithymoquinone [145, 201]. The scheme of oxidative degradation of thymoquinone is shown in Figure 2.

THY

Figure 2 — Scheme of oxidative degradation of thymoquinone and thymol

Note: TQ — thymoquinone; THQ — thymohydroquinone; DTQ — dithymoquinone; THY — thymol.

Some authors have established certain pharmacotherapeutic properties for thymohydroquinone and dithymoquinone [120, 173, 195, 196]. In addition, it is suggested that thymoquinone and the products of its degradation—dithymoquinone and thymohydroquinone—are the active constituent of Nigella sativa seeds known as nigellone [120].

In view of the above, the relevance of conducting studies that clarify the amount of thymoquinone in Nigella seeds using modern rapid, highly precise, and sensitive methods increases. This is particularly important when the quality of Nigella sativa seeds must be standardized as medicinal plant raw material according to their active substance content.

Considering the chemical composition of Nigella sativa seeds as a whole, the value of hydrophilic compounds that make a substantial contribution to the pharmacotherapeutic potential of this plant raw material cannot be overlooked. For example, researchers at a Tunisian biotechnology center established the qualitative composition [178] of phenolic compounds both in the shoots and roots of Nigella sativa, including gallic acid, p-dihydroxybenzoic acid, chlorogenic acid, p-coumaric acid, ferulic acid, vanillic acid, trans-2-hydroxycinnamic acid, trans-cinnamic acid, (-)-epicatechin, (+)-catechin hydrate, quercetin, apigenin, amentoflavone, and flavone. Quantitative determination by the authors of the sum of phenolic compounds in the shoots and roots of Nigella sativa established an average total content of 215.373 mg/100 g and 135.756 mg/100 g, respectively. In the shoots, the predominant group was phenolic carboxylic acids, among which gallic acid (27.86 mg/100 g) and vanillic acid (143.21 mg/100 g) predominated by content. Meanwhile, there are virtually no data on the qualitative and quantitative composition of phenolic compounds in Nigella seeds. This group of BAS has hepatoprotective, choleretic, and antioxidant properties [48, 89, 103].

The mineral composition of Nigella sativa seeds was determined by atomic absorption spectroscopy and consists of potassium, magnesium, calcium, sodium, phosphorus, iron, copper, zinc, and manganese. Data obtained by foreign scientists while studying macroelements made it possible to determine their content at 1.8% [172]. According to domestic researchers, the total content of macroelements was about 4.4%, and that of microelements was about 0.084% [52]. As can be seen, the results obtained are contradictory and require clarification.